Dually derivatized chitosan nanoparticles and methods of making and using the same for gene transfer in vivo

ABSTRACT

Provided herein is chitosan-derivative nanoparticle comprising chitosan functionalized with a cationic amino acid and a hydrophilic polyol; and methods of making and using same, e.g., for gene delivery in vivo.

FIELD OF INVENTION

The present invention generally relates to nanoparticles comprisingdually derivatized chitosan, and methods of making and using the samefor delivering nucleic acids, e.g., gene transfer, in vivo.

BACKGROUND OF THE INVENTION

Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine andD-glucosamine. Chitosan can form a complex with nucleic acid and, as abiocompatible and non-toxic polysaccharide, has been used as a DNAdelivery vehicle to transfect cells. Much interest has been focused onusing chitosan in non-viral delivery of nucleic acid due to thecomplexities and potential toxicity of the viral vector

A number of chitosan/DNA complexes, including complexes between modifiedchitosan and nucleic acids, have been examined in an attempt to identifycompositions well suited for gene transfection. See, e.g.,WO2010/088565; WO2008/082282. The complexes have been found to vary in,among other properties, solubility, propensity for aggregation, complexstability, particle size, ability to release DNA, and transfectionefficiency.

Thus, there is a need for new compositions and methods for gene transferin vivo with improved transfection efficiency. The compositions andmethods described herein help meet these and other needs.

SUMMARY OF INVENTION

Provided herein is the surprising discovery that chitosan functionalizedwith (1) arginine (Arg) and (2) a hydrophilic polyol (HP) at aparticular Arg:HP ratio exhibits a dramatically improved transfectionefficiency. Arginine and gluconic acid have been shown to actsynergistically as functional groups to increase the transfectionefficiency of chitosan nanoparticles. See, e.g., PCT/CA2013/050218.Disclosed herein is the surprising discovery that other molecules can besubstituted for gluconic acid as derivatives of chitosan and stilldisplay this synergistic effect. Moreover, optimized functionalizationdegree ratios of the two substituents are provided within which thederivatized chitosan exhibits maximum transfection efficiency.Accordingly provided herein are novel compositions to facilitate thedelivery of nucleic acids to cells, tissues, and organs, e.g., in vivo.In particular, provided herein are dually derivatized chitosan basednanoparticles, wherein the chitosan has been functionalized with thecationic amino acid arginine (Arg) and a hydrophilic polyol (HP) atoptimized Arg final functionalization degree:HP final functionalizationdegree (i.e., Arg:HP) ratios.

In preferred embodiment, the nanoparticles comprise chitosan that iscoupled to both a plurality of arginine and a plurality of hydrophilicpolyols, see, e.g., Formula I

wherein n is an integer of 1 to 650,α is the functionalization degree of arginine,β is the functionalization degree of the hydrophilic polyol andeach R¹ is independently selected from hydrogen, acetyl, arginine, and ahydrophilic polyol.

In a preferred embodiment, the nanoparticles comprise chitosan that iscoupled to arginine. See Formula II. In a preferred embodiment, thearginine helps to increase the solubility of chitosan and/or allows thederivatized chitosan to bind nucleic acids at a higher pH.

In another embodiment, the nanoparticles comprise chitosan that iscoupled to hydrophilic polyol, which preferably has a carboxyl oraldehyde group for coupling to chitosan. In one embodiment, thehydrophilic polyol is selected from the group consisting of a moleculehaving a carboxyl group or aldehyde group for coupling to chitosan, anda saccharide, wherein said hydrophilic polyol is not gluconic acid. Inone embodiment, the hydrophilic polyol has a carboxyl group. In oneembodiment, the hydrophilic polyol having a carboxyl group for couplingto chitosan is selected from the group consisting of gluconic acid andthreonic acid. In one embodiment, the hydrophilic polyol is gluconicacid, see, e.g., Formula III. In another embodiment, the hydrophilicpolyol is threonic acid, see, e.g., Formula IV. In another embodiment,the hydrophilic polyol is a saccharide, which may be natural orsynthetic, or the acid form of a saccharide. Nonlimiting examplesinclude glyceraldehyde, threose, erythrose, ribose, arabinose, xylose,lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, andtalose. In one embodiment, the hydrophilic polyol is glucose, see, e.g.,Formula V. In one embodiment, the hydrophilic polyol is threose, see,e.g., Formula VI.

Dually derivatized chitosan as described herein comprises arginine and ahydrophilic polyol at optimized Arg:HP final functionalization degreeratios. In a preferred embodiment, the dually derivatized chitosan has aArg:HP final functionalization degree, or molar, ratio between about 1:1and about 10:1. In another embodiment, the Arg:HP finalfunctionalization degree ratios is between about 3:1 to about 7:1. Inanother embodiment, the Arg:HP final functionalization degree ratio isabout 5:1.

In particular, chitosan-nucleic acid polyplexes formed with such duallyderivatized chitosan (“DD-chitosan”) exhibit a higher transfectionefficiency than nucleic acid polyplexes formed with non-functionalizedchitosan, singly derivatized chitosan, or chitosan dually derivatized atArg:HP final functionalization degree ratios between 1:1 and 10:1. Otherdesirable properties conferred by the use of dually functionalizedchitosan in polyplexes described herein include an improved ability topenetrate the mucous barrier, enhanced polyplex stability at a pHgreater than 6.5, reduced aggregation at a high polyplex concentrations,reduced cellular toxicity and enhanced intracellular release of nucleicacid. Further, in some preferred embodiments, the subject DD-chitosanpolyplex compositions can be administered at physiological pH (e.g.,systemic administration).

Accordingly, in one aspect, the invention provides DD-chitosan nucleicacid polyplexes. The DD-chitosan nucleic acid polyplexes comprisechitosan that is dually derivatized with arginine and a hydrophilicpolyol.

In one embodiment, the nucleic acid of the DD-chitosan nucleic acidpolyplex is DNA.

In one embodiment, the nucleic acid of the DD-chitosan nucleic acidpolyplex is RNA.

In one embodiment, the nucleic acid of the DD-chitosan nucleic acidpolyplex is an artificial nucleic acid. In a preferred embodiment, theartificial nucleic acid is selected from the group consisting of peptidenucleic acid (PNA), phosphorodiamidate morpholino oligo (PMO), lockednucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid(TNA).

In one embodiment, the nucleic acid of the DD-chitosan nucleic acidpolyplex is a therapeutic nucleic acid. In one embodiment, thetherapeutic nucleic acid is a therapeutic RNA. In a preferredembodiment, the therapeutic RNA is selected from the group consisting ofantisense RNA, siRNA, short hairpin RNA, micro RNA, and enzymatic RNA.

In one embodiment, the therapeutic nucleic acid is DNA.

In one embodiment, the therapeutic nucleic acid comprises a nucleic acidsequence encoding a therapeutic protein.

In one aspect, the invention provides a composition comprising aplurality of DD-chitosan nucleic acid polyplexes.

In one embodiment, the composition has a pH between 3.0-8.0, morepreferably between 4.0-7.0, and most preferably between 4.5-6.5.

In one aspect, the invention provides a pharmaceutical compositioncomprising a DD-chitosan nucleic acid polyplex of the invention. In apreferred embodiment, the DD-chitosan nucleic acid polyplex comprises atherapeutic nucleic acid.

In one aspect, the invention provides methods of treating disease,comprising administering a therapeutically effective amount of apharmaceutical composition of the invention to a patient.

In one embodiment, the subject pharmaceutical composition isadministered at physiological pH.

In one embodiment, the subject pharmaceutical composition isadministered systemically.

In one embodiment, the subject pharmaceutical composition isadministered locally to a target tissue. In a preferred embodiment, thesubject pharmaceutical composition is administered to mucosal tissue. Inone embodiment, the mucosal tissue is gastrointestinal (GI) tissue.

In one aspect, the invention provides a vaccine, comprising aDD-chitosan nucleic acid polyplex, wherein the nucleic acid encodes anantigen.

In one aspect, the invention provides methods for vaccinating a patient.The methods comprise administering a vaccine of the invention to apatient.

In one aspect, the invention provides an immunogenic composition,comprising a DD-chitosan nucleic acid polyplex, wherein the nucleic acidencodes an immunogen.

In one aspect, the invention provides methods for initiating orincreasing an immune response to a molecule of the interest. The methodscomprise administering an immunogenic composition of the invention to apatient, wherein the nucleic acid encodes an epitope of the molecule ofinterest. In another aspect, the invention provides methods formodulating an immune response, comprising administering achitosan-nucleic acid polyplex of the invention to a patient, whereinthe nucleic acid encodes an immunomodulatory cytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the in vitro transfection efficiency (ng SEAP/mg protein;y-axis) of 5 kDa chitosan functionalized (x-axis) with only gluconicacid at a final functionalization degree of 3% or 10%, with onlyarginine at a final functionalization degree of 10%, or with bothgluconic acid and arginine at an arginine to gluconic acid finalfunctionalization degree ratio of about 1:1 or about 3.3:1.

FIG. 2 shows the transfection efficiency (ng SEAP/mg protein; y-axis) of5 kDa chitosan functionalized (x-axis) with only gluconic acid at afinal functionalization degree of 3% to 10%, with only arginine at afinal functionalization degree of 52%, or with both gluconic acid andarginine at an arginine to gluconic acid final functionalization degreeratio of about 6:1 or about 17:1

FIG. 3 shows the transfection efficiency (ng SEAP/mg protein; y-axis) of5 kDa chitosan functionalized (x-axis) with only gluconic acid at afinal functionalization degree of 3% to 10%, with only arginine at afinal functionalization degree of 26%, or with both gluconic acid andarginine at an arginine to gluconic acid final functionalization degreeratio of about 4:1.

FIG. 4 shows the transfection efficiency (ng SEAP/mg protein; y-axis) of5 kDa chitosan functionalized (x-axis) with only threonic acid at afinal functionalization degree of 3% or, with only arginine at a finalfunctionalization degree of 29%, or with both threonic acid and arginineat an arginine to threonic acid final functionalization degree ratio ofabout 10:1.

DETAILED DESCRIPTION

Chitosan is the deacetylated form of chitin, which is a polymer ofN-acetylglucosamine that is the main component of the exoskeletons ofcrustaceans (e.g. shrimp, crab, lobster). Chitosan is formed from chitinby deacetylation, and as such is not a single polymeric molecule, but aclass of molecules having different molecular weights and differentdegrees of deacetylation. The percent deacetylation in commercialchitosans is typically between 50-100%.

The chitosan derivatives described herein are generated byfunctionalizing the resulting free amino groups with positively chargedand/or hydrophilic moieties, as described herein. The derivatizedchitosans described herein have a number of properties which areadvantageous for a nucleic acid delivery vehicle including: theyeffectively bind and complex the negatively charged nucleic acids, theycan be formed into nanoparticles of a controllable size, they can betaken up by the cells and they can release the nucleic acids at theappropriate time within the cells.

Chitosans with any degree of deacetylation (DDA) greater than 50% areused in the present invention, with functionalization between 1% and50%. (Percent functionalization is determined relative to the number offree amino moieties on the chitosan polymer.) The degrees ofdeacetylation and functionalization impart a specific charge density tothe functionalized chitosan derivative. The resulting charge densityaffects solubility, nucleic acid binding and subsequent release, andinteraction with mammalian cell membranes. Thus, in accordance with thepresent invention, these properties must be optimized for optimalefficacy. Exemplary chitosan derivatives are described in Baker et al;Ser. No. 11/657,382 filed on Jan. 24, 2007, which is incorporated hereinby reference. In one embodiment, the dually derivatized chitosandescribed herein comprises chitosan having a degree of deacetylation ofat least 50%. In one embodiment, the degree of deacetylation is at least60%, more preferably at least 70%, more preferably at least 80%, morepreferably at least 90%, and most preferably at least 95%. In apreferred embodiment, the dually derivatized chitosan described hereincomprises chitosan having a degree of deacetylation of at least 98%.

The chitosan derivatives described herein have a range of averagemolecular weights that are soluble at neutral and physiological pH, andinclude for the purposes of this invention molecular weights rangingfrom 3-110 kDa. Embodiments described herein feature lower averagemolecular weight of derivatized chitosans (<25 kDa, e.g., from about 5kDa to about 25 kDa), which can have desirable delivery and transfectionproperties, and are small in size and have favorable solubility. A loweraverage molecular weight derivatized chitosan is generally more solublethan one with a higher molecular weight, the former thus producing anucleic acid/chitosan complex that will release more easily the nucleicacid and provide increased transfection of cells. Much literature hasbeen devoted to the optimization of all of these parameters for chitosanbased delivery systems.

An ordinarily skilled artisan will recognize that chitosan refers to aplurality of molecules having a structure of Formula I, wherein n is anyinteger, and each R¹ is independently selected from acetyl or hydrogen,wherein the degree of R¹ selected from hydrogen is between 50% to 100%.Also, chitosan referred to as having an average molecular weight, e.g.,of 3 kD to 110 kD, generally refers to a plurality of chitosan moleculeshaving a weight average molecular weight of, e.g., 3 kD to 110 kD,respectively, wherein each of the chitosan molecules may have differentchain lengths (n+2). It is also well-recognized that chitosan referredto as “n-mer chitosan,” does not necessarily comprise chitosan moleculesof Formula I, wherein each chitosan molecule has a chain length of n+2.Rather, “n-mer chitosan” as used herein refers a plurality of chitosanmolecules, each of which may have different chain lengths, wherein theplurality has an average molecule weight substantially similar to orequal to a chitosan molecule having a chain length of n. For example,24-mer chitosan may comprise a plurality of chitosan molecules, eachhaving different chain lengths ranging from, e.g., 7-50, but which has aweight average molecular weight substantially similar or equivalent to achitosan molecule having a chain length of 24.

The functionalized chitosan derivatives described herein are duallyderivatized-chitosan compounds, e.g., chitosan-Arg-HP compounds. Ingeneral, the chitosan-Arg-HP compounds have the following structure ofFormula I

wherein n is an integer of 1 to 650,α is the final functionalization degree of Arg,β is the final functionalization degree of HP; andeach R¹ is independently selected from hydrogen, acetyl, an Arg, and anHP.

A dually derivatized chitosan of the invention may be functionalizedwith the cationic amino acid, arginine.

A dually derivatized chitosan of the invention may also befunctionalized with a hydrophilic polyol which may help to increase thehydrophilicity of chitosan (including Arg-chitosan) and/or may donate ahydroxyl group.

Provided are chitosan-derivative nanoparticles comprising chitosanfunctionalized with arginine (Arg) and a hydrophilic polyol (HP) ofFormula VII:

wherein:R² is selected from: H and hydroxyl;R³ is selected from: H and hydroxyl; andX is selected from: C₂-C₆ alkylene optionally substituted with one ormore hydroxyl substituents.

In some embodiments, the chitosan-derivative nanoparticle comprises ahydrophilic polyol of Formula VII:

wherein:R² is selected from: H and hydroxyl;R³ is selected from: H and hydroxyl; andX is selected from: C₂-C₆ alkylene optionally substituted with one ormore hydroxyl substituents; provided said hydrophilic polyol is notgluconic acid.

The term “C₂-C₆ alkylene” as used herein refers to a linear or brancheddivalent hydrocarbon radical optionally containing one or morecarbon-carbon multiple bonds. For the avoidance of doubt, the term“C₂-C₆ alkylene” as used herein encompasses divalent radicals ofalkanes, alkenes and alkynes.

A hydrophilic polyol according to the present invention may have a 3, 4,5, 6, or 7 carbon backbone. In one embodiment, a hydrophilic polyolaccording to the present invention having 3 to 7 carbons may have 2, 3,4, 5, or 6 hydroxyl groups. In one embodiment, a hydrophilic polyolaccording to the present invention having 3 to 7 carbons may have one ormore carbon-carbon multiple bonds. In a preferred embodiment, ahydrophilic polyol according to the present invention comprises acarboxyl group. In a further preferred embodiment, a hydrophilic polyolaccording to the present invention comprises an aldehyde group. Askilled artisan will recognize that when a hydrophilic polyol accordingto the present invention comprises an aldehyde group, such hydrophilicpolyol encompasses both the open-chain conformation (aldehyde) and thecyclic conformation (hemiacetal).

Non-limiting examples of a hydrophilic polyols includes gluconic acid,threonic acid, glucose and threose, see, e.g., Formulae III-VI. Examplesof other such hydrophilic polyols, which may have a carboxyl and/oraldehyde group, or may be a saccharide or acid form thereof, areincluded in Tables 1-3 below. A skilled artisan will recognize thatTables 1-3 provide non-limiting examples of hydrophilic polyols, andfurther, that the hydrophilic polyols shown are not limited to thestereochemistry shown.

TABLE 1 Chemical Structures of Certain Hydrophilic Polyol CarboxylicAcids and Aldehydes D-Glyceric Acid

L-Glyceric Acid

D-Threonic Acid

L-Threonic acid

D-Erythronic acid

L-Erythronic acid

D-Ribonic acid

L-Ribonic acid

D-Arabonic acid

L-Arabonic acid

D-Xylonic acid

L-Xylonic acid

D-Lyxonic acid

L-Lyxonic acid

D-Allonic acid

L-Allonic acid

D-Gluconic acid

L-Gluconic acid

D-Altronic acid

L-Altronic acid

D-Mannonic acid

L-Mannonic acid

D-Gulonic acid

L-Gulonic acid

D-Idonic acid

L-Idonic acid

D-Galactonic acid

L-Galactonic acid

D-Talonic acid

L-Talonic acid

D-Fuconic Acid

L-Fuconic Acid

L-Glycero-D- Mannoheptonic Acid

L-Glycero-D- Mannoheptonic Acid

D-Glyceraldehyde

L-Glyceraldehyde

D-Threose

L-Threose

D-Erythrose

L-Erythrose

D-Ribose

L-Ribose

D-Arabose

L-Arabose

D-Xylose

L-Xylose

D-Lyxose

L-Lyxose

D-Allose

L-Allose

D-Glucose

L-Glucose

D-Altrose

L-Altrose

D-Mannose

L-Mannose

D-Gulose

L-Gulose

D-Idose

L-Idose

D-Galactose

L-Galactose

D-Talose

L-Talose

D-Fucose

L-Fucose

L-Glycero-D- Mannoheptose

D-Glycero-L- Mannoheptose

TABLE 2 Chemical Names of Certain Hydrophilic Polyol Carboxylic AcidsNumber of Number of Carbons hydroxyls IUPAC Name 7 carbons 6 hydroxyls2,3,4,5,6,7-hexahydroxylheptanoic acid 5 hydroxyls2,3,4,5,7-pentahydroxylheptanoic acid 2,3,4,6,7-pentahydroxylheptanoicacid 2,3,5,6,7-pentahydroxylheptanoic acid2,4,5,6,7-pentahydroxylheptanoic acid 3,4,5,6,7-pentahydroxylheptanoicacid 4 hydroxyls 2,3,4,5-tetrahydroxylheptanoic acid2,3,4,6-tetrahydroxylheptanoic acid 2,3,4,7-tetrahydroxylheptanoic acid2,3,5,6-tetrahydroxylheptanoic acid 2,3,5,7-tetrahydroxylheptanoic acid2,3,6,7-tetrahydroxylheptanoic acid 2,3,6,7-tetrahydroxylheptanoic acid2,4,5,6-tetrahydroxylheptanoic acid 2,4,5,7-tetrahydroxylheptanoic acid2,4,6,7-tetrahydroxylheptanoic acid 2,5,6,7-tetrahydroxylheptanoic acid2,5,6,7-tetrahydroxylheptanoic acid 3,4,5,6-tetrahydroxylheptanoic acid3,4,5,7-tetrahydroxylheptanoic acid 3,4,6,7-tetrahydroxylheptanoic acid3,5,6,7-tetrahydroxylheptanoic acid 4,5,6,7-tetrahydroxylheptanoic acid3 hydroxyls 2,3,4-trihydroxylheptanoic acid 2,3,5-trihydroxylheptanoicacid 2,3,6-trihydroxylheptanoic acid 2,3,7-trihydroxylheptanoic acid2,4,5-trihydroxylheptanoic acid 2,4,6-trihydroxylheptanoic acid2,5,6-trihydroxylheptanoic acid 2,5,7-trihydroxylheptanoic acid2,6,7-trihydroxylheptanoic acid 3,4,5-trihydroxylheptanoic acid3,4,6-trihydroxylheptanoic acid 3,5,6-trihydroxylheptanoic acid4,5,6-trihydroxylheptanoic acid 4,5,7-trihydroxylheptanoic acid5,6,7-trihydroxylheptanoic acid 2 hydroxyls 2,3-dihydroxylheptanoic acid2,4-dihydroxylheptanoic acid 2,5-dihydroxylheptanoic acid2,6-dihydroxylheptanoic acid 2,7-dihydroxylheptanoic acid3,4-dihydroxylheptanoic acid 3,5-dihydroxylheptanoic acid3,6-dihydroxylheptanoic acid 3,7-dihydroxylheptanoic acid4,5-dihydroxylheptanoic acid 4,6-dihydroxylheptanoic acid4,7-dihydroxylheptanoic acid 5,6-dihydroxylheptanoic acid5,7-dihydroxylheptanoic acid 6,7-dihydroxylheptanoic acid 6 carbons 5hydroxyls 2,3,4,5,6-pentahydroxylhexanoic acid 4 hydroxyls2,3,4,5-tetrahydroxylhexanoic acid 2,3,4,6-tetrahydroxylhexanoic acid2,3,5,6-tetrahydroxylhexanoic acid 2,4,5,6-tetrahydroxylhexanoic acid3,4,5,6-tetrahydroxylhexanoic acid 3 hydroxyls 4,5,6-trihydroxylhexanoicacid 3,4,5-trihydroxylhexanoic acid 2,3,4-trihydroxylhexanoic acid3,4,6-trihydroxylhexanoic acid 2,3,6-trihydroxylhexanoic acid1,3,6-trihydroxylhexanoic acid 1,5,6-trihydroxylhexanoic acid2,5,6-trihydroxylhexanoic acid 2,3,5-trihydroxylhexanoic acid2,4,5-trihydroxylhexanoic acid 2 hydroxyls 5,6-dihydroxylhexanoic acid4,6-dihydroxylhexanoic acid 3,6-dihydroxylhexanoic acid2,6-dihydroxylhexanoic acid 4,5-dihydroxylhexanoic acid3,5-dihydroxylhexanoic acid 2,5-dihydroxylhexanoic acid3,4-dihydroxylhexanoic acid 2,4-dihydroxylhexanoic acid2,3-dihydroxylhexanoic acid 5 carbons 4 hydroxyls2,3,4,5-tetrahydroxylpentanoic acid 3 hydroxyls3,4,5-trihydroxylpentanoic acid 2,3,5-trihydroxylpentanoic acid1,4,5-trihydroxylpentanoic acid 2,4,5-trihydroxylpentanoic acid2,3,5-trihydroxylpentanoic acid 2,3,4-trihydroxylpentanoic acid 2hydroxyls 4,5-dihydroxylpentanoic acid 3,5-dihydroxylpentanoic acid2,5-dihydroxylpentanoic acid 3,4-dihydroxylpentanoic acid2,4-dihydroxylpentanoic acid 2,3-dihydroxylpentanoic acid 4 carbons 3hydroxyls 2,3,4-trihydroxylbutanoic acid 2 hydroxyls3,4-dihydroxylbutanoic acid 2,4-dihydroxylbutanoic acid2,3-dihydroxylbutanoic acid 3 carbons 2 hydroxyls2,3-dihydroxylpropanoic acid

TABLE 3 Chemical Names of Certain Hydrophilic Polyol Aldehydes Number ofNumber of Carbons polyols IUPAC Name 7 carbons 6 hydroxyls2,3,4,5,6,7-hexahydroxylheptanal 5 hydroxyls2,3,4,5,7-pentahydroxylheptanal 2,3,4,6,7-pentahydroxylheptanal2,3,5,6,7-pentahydroxylheptanal 2,4,5,6,7-pentahydroxylheptanal3,4,5,6,7-pentahydroxylheptanal 4 hydroxyls2,3,4,5-tetrahydroxylheptanal 2,3,4,6-tetrahydroxylheptanal2,3,4,7-tetrahydroxylheptanal 2,3,5,6-tetrahydroxylheptanal2,3,5,7-tetrahydroxylheptanal 2,3,6,7-tetrahydroxylheptanal2,3,6,7-tetrahydroxylheptanal 2,4,5,6-tetrahydroxylheptanal2,4,5,7-tetrahydroxylheptanal 2,4,6,7-tetrahydroxylheptanal2,5,6,7-tetrahydroxylheptanal 2,5,6,7-tetrahydroxylheptanal3,4,5,6-tetrahydroxylheptanal 3,4,5,7-tetrahydroxylheptanal3,4,6,7-tetrahydroxylheptanal 3,5,6,7-tetrahydroxylheptanal4,5,6,7-tetrahydroxylheptanal 3 hydroxyls 2,3,4-trihydroxylheptanal2,3,5-trihydroxylheptanal 2,3,6-trihydroxylheptanal2,3,7-trihydroxylheptanal 2,4,5-trihydroxylheptanal2,4,6-trihydroxylheptanal 2,5,6-trihydroxylheptanal2,5,7-trihydroxylheptanal 2,6,7-trihydroxylheptanal3,4,5-trihydroxylheptanal 3,4,6-trihydroxylheptanal3,5,6-trihydroxylheptanal 4,5,6-trihydroxylheptanal4,5,7-trihydroxylheptanal 5,6,7-trihydroxylheptanal 2 hydroxyls2,3-dihydroxylheptanal 2,4-dihydroxylheptanal 2,5-dihydroxylheptanal2,6-dihydroxylheptanal 2,7-dihydroxylheptanal 3,4-dihydroxylheptanal3,5-dihydroxylheptanal 3,6-dihydroxylheptanal 3,7-dihydroxylheptanal4,5-dihydroxylheptanal 4,6-dihydroxylheptanal 4,7-dihydroxylheptanal5,6-dihydroxylheptanal 5,7-dihydroxylheptanal 6,7-dihydroxylheptanal 6carbons 5 hydroxyls 2,3,4,5,6-pentahydroxylhexanal 4 hydroxyls2,3,4,5-tetrahydroxylhexanal 2,3,4,6-tetrahydroxylhexanal2,3,5,6-tetrahydroxylhexanal 2,4,5,6-tetrahydroxylhexanal3,4,5,6-tetrahydroxylhexanal 3 hydroxyls 4,5,6-trihydroxylhexanal3,4,5-trihydroxylhexanal 2,3,4-trihydroxylhexanal3,4,6-trihydroxylhexanal 2,3,6-trihydroxylhexanal1,3,6-trihydroxylhexanal 1,5,6-trihydroxylhexanal2,5,6-trihydroxylhexanal 2,3,5-trihydroxylhexanal2,4,5-trihydroxylhexanal 2 hydroxyls 5,6-dihydroxylhexanal4,6-dihydroxylhexanal 3,6-dihydroxylhexanal 2,6-dihydroxylhexanal4,5-dihydroxylhexanal 3,5-dihydroxylhexanal 2,5-dihydroxylhexanal3,4-dihydroxylhexanal 2,4-dihydroxylhexanal 2,3-dihydroxylhexanal 5carbons 4 hydroxyls 2,3,4,5-tetrahydroxylpentanal 3 hydroxyls3,4,5-trihydroxylpentanal 2,3,5-trihydroxylpentanal1,4,5-trihydroxylpentanal 2,4,5-trihydroxylpentanal2,3,5-trihydroxylpentanal 2,3,4-trihydroxylpentanal 2 hydroxyls4,5-dihydroxylpentanal 3,5-dihydroxylpentanal 2,5-dihydroxylpentanal3,4-dihydroxylpentanal 2,4-dihydroxylpentanal 2,3-dihydroxylpentanal 4carbons 3 hydroxyls 2,3,4-trihydroxylbutanal 2 hydroxyls3,4-dihydroxylbutanal 2,4-dihydroxylbutanal 2,3-dihydroxylbutanal 3carbons 2 hydroxyls 2,3-dihydroxylpropanal

In a preferred embodiment, the HP may be selected from the groupconsisting of 2,3-dihydroxylpropanoic acid;2,3,4,5,6,7-hexahydroxylheptanal; 2,3,4,5,6-pentahydroxylhexanal;2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal.

In a preferred embodiment, the HP may be selected from the groupconsisting of D-glyceric acid, L-glyceric acid,L-glycero-D-mannoheptose, D-glycero-L-mannoheptose, D-glucose,L-glucose, D-fucose, L-fucose, D-glyceraldehyde, and L-glyceraldehyde.

In a preferred embodiment, the α:β ratio is between about 1:1 and about10:1. In a preferred embodiment, the ratio is between about 3:1 andabout 7:1. In a most preferred embodiment, the ratio is about 5:1.

A non-limiting method for conjugating chitosan with arginine or ahydrophilic polyol in an aqueous medium, in accordance with the presentinvention, is described herein. The method utilizes well-known watersoluble 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS) to catalyze the formation of amide between anamine on the chitosan backbone and a carboxylic acid on a Boc-protectedArg or a hydrophilic polyol having a carboxyl group.

Generally, chitosan in dilute HCl solution with an adjusted pH for atargeted coupling pH of, e.g., 6.0±0.5 and more preferably 6.0±0.2, isfirst coupled to either Arg, e.g., Boc-Arg or hydrophilic polyol havinga carboxyl group, purified, and then coupled with the second functionalgroup. For example, if chitosan is first coupled to a Arg, theArg-coupled chitosan (Arg-chitosan) may be purified and then coupled tothe hydrophilic polyol. Conversely, if chitosan is first coupled to thehydrophilic polyol, the hydrophilic polyol-coupled chitosan may bepurified and then coupled to an Arg. Irrespective of the order ofcoupling, the Arg and hydrophilic polyol may be coupled to chitosanusing well-known methods.

For example, a Arg may be coupled to chitosan or a polyol-functionalizedchitosan (polyol-chitosan) by adding a mixture of Boc-Arg and NHSaqueous solution of adjusted pH into chitosan in dilute HCl followed byadding EDC water solution to initiate coupling at room temperature for24 hours. The concentration of chitosan amine, reaction pH and the molarratios of Arg-COOH over chitosan-amine and EDC:NHS:Arg-COOH may bepre-calculated and satisfied to have reproducible finalfunctionalization degree of Arg. Boc-Arg-chitosan may be purified priorto the De-Boc reaction. De-Boc may proceed in HCl medium with acontrolled HCl concentration and reaction time. Any depolymerization ofchitosan during de-Boc may be monitored by measuring the change inviscosity of the reaction solution, which was proven to be negligible,and the efficiency of de-Boc may be ascertained by Nuclear MagneticResonance (NMR) methods on de-Boc-Arg-chitosan and Boc-Arg-chitosan. Thefunctionalization degree may be determined from C, N elemental analysisof the purified de-Boc-Arg-chitosan.

A hydrophilic polyol that has a carboxyl group may be coupled tochitosan or Arg-coupled chitosan (Arg-chitosan) at a reaction pH of6.0±0.3. At this pH, the carboxylic acid group of the hydrophilic polyolmay be attacked by uncoupled amines on the chitosan backbone accordingto a nucleophilic substitution reaction mechanism. An ordinarily skilledartisan will recognize that, when coupling such a hydrophilic polyol toArg-chitosan, it is also possible that a small amount of the hydrophilicpolyol may form a covalent bond with an amine group of the Arg throughthe same mechanism, although it is likely that the nucleophilicsubstitution reaction will occur predominantly with the amine group ofthe chitosan backbone.

A hydrophilic polyol that is a natural saccharide may be coupled tochitosan or Arg-coupled chitosan (Arg-chitosan) using reductiveamination followed by reduction with NaCNBH₃ or NaBH₄.

Boc-Arg-chitosan, de-Boc-Arg-chitosan, polyol-chitosan, and/or duallyderivatized chitosan may be purified via precipitation, or columntreatment, or regular dialysis, or inverse-flow dialysis against waterusing dialysis tubing of appropriate molecular weight cut off (MWCO), orthrough a tangential-flow-filtration (TFF) and diafiltration cartridges.

Accordingly, “dually derivatized-chitosan” or “DD-chitosan” also refersto chitosan that has been dually functionalized (“duallyfunctionalized-chitosan” or “DF-chitosan), e.g., coupled with both a Argand a hydrophilic polyol, both of which are covalently attached tochitosan. The Arg may be covalently attached to chitosan either assingle amino acid or as a polypeptide.

As used herein, unless otherwise indicated, the term “peptide” and“polypeptide” are used interchangeably.

The term “polypeptide” is used in its broadest sense to refer toconventional polypeptides (i.e., short polypeptides containing L orD-amino acids), as well as peptide equivalents, peptide analogs andpeptidomimetics that retain the desired functional activity. Peptideequivalents can differ from conventional peptides by the replacement ofone or more amino acids with related organic acids, amino acids or thelike, or the substitution or modification of side chains or functionalgroups.

Peptidomimetics may have one or more peptide linkages replaced by analternative linkage, as is known in the art. Portions or all of thepeptide backbone can also be replaced by conformationally constrainedcyclic alkyl or aryl substituents to restrict mobility of the functionalamino acid sidechains, as is known in the art.

The polypeptides of this invention may be produced by recognizedmethods, such as recombinant and synthetic methods that are well knownin the art. Techniques for the synthesis of peptides are well known andinclude those described in Merrifield, J. Amer. Chem. Soc. 85:2149-2456(1963), Atherton, et al., Solid Phase Peptide Synthesis: A PracticalApproach, IRL Press (1989), and Merrifield, Science 232:341-347 (1986).

As used herein, “linear polypeptide” refers to a polypeptide that lacksbranching groups covalently attached to its constituent amino acid sidechains. As used herein, “branched polypeptide” refers to a polypeptidethat comprises branching groups covalently attached to its constituentamino acid side chains.

As stated above, the Arg final functionalization degree or polyol finalfunctionalization degree of a DD-chitosan of the invention may bedetermined by elemental analysis. The following describes an exemplarycalculation of the final functionalization degree of, e.g., arginine andgluconic acid that are coupled to chitosan backbone. For clarity, thefollowing example uses a 20-mer chitosan molecule of 100% DDA.

Chitosan of 100% DDA is a homopolymer of D-glucosamine; this repeat unithas 6 C atoms and 1 N atom so that the molar ratio of C/N is 6/1.

Due to the depolymerization process, the terminal unit at one end of thepolymer is a 2,5-anhydro-D-mannose. This unit has a C/N ratio of 6/0.

Consequently, in the 20-mer chitosan example, there are 19 units ofD-glucosamine and 1 unit of 2,5-anhydro-D-mannose. Therefore, the totalratio of C/N is:

$\frac{C}{N} = {\frac{\left( {19 \times 6} \right) + \left( {1 \times 6} \right)}{\left( {19 \times 1} \right) + \left( {1 \times 0} \right)} = \frac{120}{19}}$

This ratio is normalized to 6 carbons (which is the average number of Catoms per unit) over a known integral number of nitrogen on average:

$\frac{120}{19} = \frac{6}{0.95}$

In coupling, e.g., arginine, to chitosan, the arginine moiety has 6 Cand 4 N. For an arginine-modified chitosan of 20 repeat units having theaverage arginine functionalization degree of R %, the followingrelationship describes the contribution of C and N from chitosan andarginine:

$\frac{C}{N} = \frac{6 + {6\; R\mspace{14mu} \%}}{0.95 + {4\; R\mspace{14mu} \%}}$

The molar ratio of C/N is calculated from the experimentally derivedmass ratio of C/N. Consequently, the functionalization degree of R %,i.e., the percentage of amino groups on the chitosan backbone that iscoupled with an arginine group, can be derived once the C/N molar ratiois known.

Coupling gluconic acid to chitosan:

The gluconic acid moiety has 6 C and 0 N. For a gluconic acid-modifiedchitosan of 20 repeat units having a functionalization degree of G %,the following relationship describes the contribution of C and N fromchitosan and gluconic acid:

$\frac{C}{N} = \frac{6 + {6G\mspace{14mu} \%}}{0.95}$

The molar ratio of C/N is calculated from the experimentally derivedmass ratio of C/N. Consequently, the functionalization degree of G %,i.e., the percentage of amine groups on the chitosan backbone that iscoupled with a gluconic acid moiety, can be derived once the C/N molarratio is known.

Coupling arginine to chitosan, followed by gluconic acid coupling:

When coupling gluconic acid to R-chitosan of 20 repeat units with apreviously determined arginine final functionalization degree (R %), thefollowing relationship describes the contribution of C and N fromchitosan, arginine and gluconic acid:

$\frac{C}{N} = \frac{6 + {6\; R\mspace{14mu} \%} + {6G\mspace{14mu} \%}}{0.95 + {4\; R\mspace{14mu} \%}}$

The molar ratio of C/N is calculated from the experimentally derivedmass ratio of C/N. Consequently, since R % was determined previously,the functionalization degree of G % can be derived once the C/N molarratio is known.

As described above, the “final functionalization degree” of Arg or HP asused herein refers to the percentage of amino groups on the chitosanbackbone functionalized with Arg or HP, respectively. Accordingly, “α:βratio”, “final functionalization degree ratio” (e.g., Arg finalfunctionalization degree: HP final functionalization degree ratio) andthe like may be used interchangeably with the term “molar ratio” or“number ratio.

In one embodiment, the dually derived chitosan is not achitosan-derivative nanoparticle or chitosan-derivative nanoparticlepolyplex as exemplified in PCT Application No. PCT/CA2013/050218,particularly in the Examples of PCT/CA2013/050218. In one embodiment,the chitosan-derivative nanoparticle is not one of the followingnanoparticles:

a. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 52% and gluconic acid at a finalfunctionalization degree of 8%b. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 3%;c. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;d. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 6%;e. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith coupled with arginine at a final functionalization degree of 26%and gluconic acid at a final functionalization degree of 9%;f. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 40, wherein the nanoparticle comprises 24 mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;g. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 10, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;h. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 26% and gluconic acid at a finalfunctionalization degree of 5%;i. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 26% and gluconic acid at a finalfunctionalization degree of 6%; orj. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 52% and gluconic acid at a finalfunctionalization degree of 8%.

In one embodiment, the chitosan-derivative nanoparticle compriseschitosan coupled with gluconic acid at a final functionalization degreeof 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or greater. In oneembodiment, the chitosan derivative nanoparticle comprises chitosancoupled with arginine at a final functionalization degree of from about1% to about 25%. In another embodiment, the chitosan derivativenanoparticle comprises chitosan coupled with arginine at a finalfunctionalization degree of from about 27% to about 51%. In oneembodiment, the chitosan derivative nanoparticle comprises chitosancoupled with arginine at a final functionalization degree of from about53% to about 70% of arginine. In another embodiment, the chitosanderivative nanoparticle comprises chitosan coupled with arginine at afinal functionalization degree which is not 26% or 52%.

In one embodiment, the chitosan derivative nanoparticle compriseschitosan coupled with arginine at a final functionalization degree of26% and coupled with gluconic acid at a final functionalization degreewhich is not 3%.

In one embodiment, the chitosan derivative nanoparticle compriseschitosan coupled with arginine at a final functionalization degree of26% and coupled with gluconic acid at a final functionalization degreewhich is not 5%. In one embodiment, the chitosan derivative nanoparticlecomprises chitosan coupled with arginine at a final functionalizationdegree of 26% and coupled with gluconic acid at a finalfunctionalization degree which is not 6%. In one embodiment, thechitosan derivative nanoparticle comprises chitosan coupled witharginine at a final functionalization degree of 26% and coupled withgluconic acid at a final functionalization degree which is not 9%. Inone embodiment, the chitosan derivative nanoparticle comprises chitosancoupled with arginine at a final functionalization degree of 26% andcoupled with gluconic acid at a final functionalization degree which isnot 3%, 5%, 6% or 9%. In one embodiment, the chitosan derivativenanoparticle comprises chitosan coupled with arginine at a finalfunctionalization degree of 52% and coupled with gluconic acid at afinal functionalization degree which is not 8%. In one embodiment, thechitosan derivative nanoparticle comprises chitosan coupled withgluconic acid at a final functionalization degree selected from 3%, 5%,6% and 9% and coupled with arginine at a final functionalization degreewhich is not 26%. In one embodiment, the chitosan derivativenanoparticle comprises chitosan coupled with gluconic acid at a finalfunctionalization degree of 8% and coupled with arginine at a finalfunctionalization degree which is not 52%.

In some embodiments, where appropriate, DD-chitosan includes DD-chitosanderivatives, e.g., DD chitosan that incorporate an additionalfunctionalization, e.g., DD-chitosan with an attached ligand.“Derivatives” will be understood to include the broad category ofchitosan-based polymers comprising covalently modifiedN-acetyl-D-glucosamine and/or D-glucosamine units, as well aschitosan-based polymers incorporating other units, or attached to othermoieties. Derivatives are frequently based on a modification of thehydroxyl group or the amine group of glucosamine, such as done witharginine-functionalized chitosan. Examples of chitosan derivativesinclude, but are not limited to, trimethylated chitosan, PEGylatedchitosan, thiolated chitosan, galactosylated chitosan, alkylatedchitosan, PEI-incorporated chitosan, uronic acid modified chitosan,glycol chitosan, and the like. For further teaching on chitosanderivatives, see, for example, pp. 63-74 of “Non-viral Gene Therapy”, K.Taira, K. Kataoka, T. Niidome (editors), Springer-Verlag Tokyo, 2005,ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007,vol. 52 (23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55(2004) 77-93.

Dispersed systems consist of particulate matter, known as the dispersedphase, distributed throughout a continuous medium. A “dispersion” ofDD-chitosan nucleic acid polyplexes is a composition comprising hydratedDD-chitosan nucleic acid polyplexes, wherein polyplexes are distributedthroughout the medium.

As used herein, a “pre-concentrated” dispersion is one that has notundergone the concentrating process to form a concentrated dispersion.

As used herein, “substantially free” of polyplex precipitate means thatthe composition is essentially free from particles that can be observedon visual inspection.

As used herein, physiological pH refers to a pH between 6 to 8.

By “DD-chitosan nucleic acid polyplex” or its grammatical equivalents ismeant a complex comprising a plurality of DD-chitosan molecules and aplurality of nucleic acid molecules. In a preferred embodiment, thedually derivatized-chitosan is complexed with said nucleic acid.

The DD-chitosan nucleic acid polyplexes comprise a nucleic acidcomponent and a DD-chitosan component. Chitosan, and DD-chitosan nucleicacid polyplexes may be prepared by any method known in the art. Forexample, functionalized chitosan and nucleotide feedstock concentrationsmay be adjusted to accommodate various amine-to-phosphate ratios (N/P),mixing ratios and target nucleotide concentrations. In some embodiments,particularly small batches, e.g., batches under 2 mL, the functionalizedchitosan and nucleotide feedstocks may be mixed by slowly dripping thenucleotide feedstock into the functionalized chitosan feedstock whilevortexing the container. In other embodiments, the functionalizedchitosan and nucleotide feedstocks may be mixed by in-line mixing thetwo fluid streams. In other embodiments, the resulting polyplexdispersion may be concentrated by TFF. A preferred method for polyplexformation is disclosed in WO 2009/039657, which is expresslyincorporated herein in its entirety by reference.

A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases nucleic acid analogs areincluded that may have alternate backbones or other modifications ormoieties incorporated for any of a variety of purposes, e.g., stabilityand protection. Other analog nucleic acids contemplated include thosewith non-ribose backbones. In addition, mixtures of naturally occurringnucleic acids, analogs, and both can be made. The nucleic acids may besingle stranded or double stranded or contain portions of both doublestranded or single stranded sequence. Nucleic acids include but are notlimited to DNA, RNA and hybrids where the nucleic acid contains anycombination of deoxyribo- and ribo-nucleotides, and any combination ofbases, including uracil, adenine, thymine, cytosine, guanine, inosine,xanthanine, hypoxanthanine, isocytosine, isoguanine, etc. Nucleic acidsinclude DNA in any form, RNA in any form, including triplex, duplex orsingle-stranded, anti-sense, siRNA, ribozymes, deoxyribozymes,polynucleotides, oligonucleotides, chimeras, microRNA, and derivativesthereof. Nucleic acids include artificial nucleic acids, including butnot limited to, peptide nucleic acid (PNA), phosphorodiamidatemorpholino oligo (PMO), locked nucleic acid (LNA), glycol nucleic acid(GNA) and threose nucleic acid (TNA).

In one embodiment, the nucleic acid component comprises a therapeuticnucleic acid. The subject DD-chitosan nucleic acid polyplexes areamenable to the use of any therapeutic nucleic acid known in the art.Therapeutic nucleic acids include therapeutic RNAs, which are RNAmolecules capable of exerting a therapeutic effect in a mammalian cell.Therapeutic RNAs include, but are not limited to, antisense RNAs,siRNAs, short hairpin RNAs, micro RNAs, and enzymatic RNAs. Therapeuticnucleic acids include, but are not limited to, nucleic acids intended toform triplex molecules, protein binding nucleic acids, ribozymes,deoxyribozymes, and small nucleotide molecules.

Many types of therapeutic RNAs are known in the art. For example, seeGrimm et al., Therapeutic application of RNAi is mRNA targeting finallyready for prime time? J. Clin. Invest., 117:3633-3641, 2007; Aagaard etal., RNAi therapeutics: Principles, prospects and challenges, Adv. DrugDeliv. Rev., 59:75-86, 2007; Dorsett et al., siRNAs: Applications infunctional genomics and potential as therapeutics, Nat. Rev. DrugDiscov., 3:318-329, 2004. These include double-stranded shortinterfering RNA (siRNA).

Therapeutic nucleic acids also include nucleic acids encodingtherapeutic proteins, including cytotoxic proteins and prodrugs.

In a preferred embodiment, the nucleic acid component comprises atherapeutic nucleic acid construct. The therapeutic nucleic acidconstruct is a nucleic acid construct capable of exerting a therapeuticeffect. Therapeutic nucleic acid constructs may comprise nucleic acidsencoding therapeutic proteins, as well as nucleic acids that producetranscripts that are therapeutic RNAs. A therapeutic nucleic acid may beused to effect genetic therapy by serving as a replacement orenhancement for a defective gene or to compensate for lack of aparticular gene product, by encoding a therapeutic product. Atherapeutic nucleic acid may also inhibit expression of an endogenousgene. A therapeutic nucleic acid may encode all or a portion of atranslation product, and may function by recombining with DNA alreadypresent in a cell, thereby replacing a defective portion of a gene. Itmay also encode a portion of a protein and exert its effect by virtue ofco-suppression of a gene product. In a preferred embodiment, thetherapeutic nucleic acid is selected from those disclosed in U.S. Ser.No. 11/694,852, which is expressly incorporated herein by reference.

In a preferred embodiment, the therapeutic nucleic acid encodes atherapeutic protein that is selected from the group consisting ofhormones, enzymes, cytokines, chemokines, antibodies, mitogenic factors,growth factors, differentiation factors, factors influencingangiogenesis, factors influencing blood clot formation, factorsinfluencing blood glucose levels, factors influencing glucosemetabolism, factors influencing lipid metabolism, factors influencingblood cholesterol levels, factors influencing blood LDL or HDL levels,factors influencing cell apoptosis, factors influencing food intake,factors influencing energy expenditure, factors influencing appetite,factors influencing nutrient absorption, factors influencinginflammation, and factors influencing bone formation. Particularlypreferred are therapeutic nucleic acids encoding insulin, leptin,glucagon antagonist, GLP-1, GLP-2, Ghrelin, cholecystokinin, growthhormone, clotting factors, PYY, erythropoietin, inhibitors ofinflammation, IL-10, IL-17 antagonists, TNFα antagonists, growth hormonereleasing hormone, or parathyroid hormone.

Expression Control Regions

In a preferred embodiment, a polyplex of the invention comprises atherapeutic nucleic acid, which is a therapeutic construct, comprisingan expression control region operably linked to a coding region. Thetherapeutic construct produces therapeutic nucleic acid, which may betherapeutic on its own, or may encode a therapeutic protein.

In some embodiments, the expression control region of a therapeuticconstruct possesses constitutive activity. In a number of preferredembodiments, the expression control region of a therapeutic constructdoes not have constitutive activity. This provides for the dynamicexpression of a therapeutic nucleic acid. By “dynamic” expression ismeant expression that changes over time. Dynamic expression may includeseveral such periods of low or absent expression separated by periods ofdetectable expression. In a number of preferred embodiments, thetherapeutic nucleic acid is operably linked to a regulatable promoter.This provides for the regulatable expression of therapeutic nucleicacids.

Expression control regions comprise regulatory polynucleotides(sometimes referred to herein as elements), such as promoters andenhancers, which influence expression of an operably linked therapeuticnucleic acid.

Expression control elements included herein can be from bacteria, yeast,plant, or animal (mammalian or non-mammalian). Expression controlregions include full-length promoter sequences, such as native promoterand enhancer elements, as well as subsequences or polynucleotidevariants that retain all or part of full-length or non-variant function(e.g., retain some amount of nutrient regulation or cell/tissue-specificexpression). As used herein, the term “functional” and grammaticalvariants thereof, when used in reference to a nucleic acid sequence,subsequence or fragment, means that the sequence has one or morefunctions of native nucleic acid sequence (e.g., non-variant orunmodified sequence). As used herein, the term “variant” means asequence substitution, deletion, or addition, or other modification(e.g., chemical derivatives such as modified forms resistant tonucleases).

As used herein, the term “operable linkage” refers to a physicaljuxtaposition of the components so described as to permit them tofunction in their intended manner. In the example of an expressioncontrol element in operable linkage with a nucleic acid, therelationship is such that the control element modulates expression ofthe nucleic acid. Typically, an expression control region that modulatestranscription is juxtaposed near the 5′ end of the transcribed nucleicacid (i.e., “upstream”). Expression control regions can also be locatedat the 3′ end of the transcribed sequence (i.e., “downstream”) or withinthe transcript (e.g., in an intron). Expression control elements can belocated at a distance away from the transcribed sequence (e.g., 100 to500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleicacid). A specific example of an expression control element is apromoter, which is usually located 5′ of the transcribed sequence.Another example of an expression control element is an enhancer, whichcan be located 5′ or 3′ of the transcribed sequence, or within thetranscribed sequence.

Some expression control regions confer regulatable expression to anoperatably linked therapeutic nucleic acid. A signal (sometimes referredto as a stimulus) can increase or decrease expression of a therapeuticnucleic acid operatably linked to such an expression control region.Such expression control regions that increase expression in response toa signal are often referred to as inducible. Such expression controlregions that decrease expression in response to a signal are oftenreferred to as repressible. Typically, the amount of increase ordecrease conferred by such elements is proportional to the amount ofsignal present; the greater the amount of signal, the greater theincrease or decrease in expression.

Numerous regulatable promoters are known in the art. Preferred inducibleexpression control regions include those comprising an induciblepromoter that is stimulated with a small molecule chemical compound. Inone embodiment, an expression control region is responsive to a chemicalthat is orally deliverable but not normally found in food. Particularexamples can be found, for example, in U.S. Pat. Nos. 5,989,910;5,935,934; 6,015,709; and 6,004,941.

In one embodiment, the therapeutic construct further comprises anintegration sequence. In one embodiment, the therapeutic constructcomprises a single integration sequence. In another embodiment, thetherapeutic construct comprises a first and a second integrationsequence for integrating the therapeutic nucleic acid or a portionthereof into the genome of a target cell. In a preferred embodiment, theintegration sequence(s) is functional in combination with a means forintegration that is selected from the group consisting of mariner,sleeping beauty, FLP, Cre, ΦC31, R, lambda, and means for integrationfrom integrating viruses such as AAV, retroviruses, and lentiviruses.

In one embodiment, the subject composition further comprises anon-therapeutic construct in addition to a therapeutic construct,wherein the non-therapeutic construct comprises a nucleic acid sequenceencoding a means for integration operably linked to a second expressioncontrol region. This second expression control region and the expressioncontrol region operably linked to the therapeutic nucleic acid may bethe same or different. The encoded means for integration is preferablyselected from the group consisting of mariner, sleeping beauty, FLP,Cre, ΦC31, R, lambda, and means for integration from integrating virusessuch as AAV, retroviruses, and lentiviruses.

For further teaching, see WO2008020318, which is expressly incorporatedherein in its entirety by reference. In one embodiment, the nucleic acidof the DD-chitosan nucleic acid polyplex is an artificial nucleic acid.

Preferred artificial nucleic acids include, but are not limited to,peptide nucleic acid (PNA), phosphorodiamidate morpholino oligo (PMO),locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleicacid (TNA).

In one embodiment, the nucleic acid of the DD-chitosan nucleic acidpolyplex is a therapeutic nucleic acid. In one embodiment, thetherapeutic nucleic acid is a therapeutic RNA. Preferred therapeuticRNAs include, but are not limited to, antisense RNA, siRNA, shorthairpin RNA, micro RNA, and enzymatic RNA.

In one embodiment, the therapeutic nucleic acid is DNA.

In one embodiment, the therapeutic nucleic acid comprises a nucleic acidsequence encoding a therapeutic protein.

In preferred embodiments, the dually derivatized (DD) chitosan nucleicacid polyplex is not a polyplex which comprises a nucleic acid whichencodes for secreted alkaline phosphatase (SEAP) or luciferase complexedto one of the following chitosan-derivative nanoparticles:

a. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 52% and gluconic acid at a finalfunctionalization degree of 8%;b. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 3%;c. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;d. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 6%;e. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 20, wherein the nanoparticle comprises 24mer chitosan coupledwith coupled with arginine at a final functionalization degree of 26%and gluconic acid at a final functionalization degree of 9%;f. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 40, wherein the nanoparticle comprises 24 mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;g. a chitosan-derivative nanoparticle having an amine/phosphate (N/P)ratio of 10, wherein the nanoparticle comprises 24mer chitosan coupledwith arginine at a final functionalization degree of 26% and gluconicacid at a final functionalization degree of 5%;h. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 26% and gluconic acid at a finalfunctionalization degree of 5%;i. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 26% and gluconic acid at a finalfunctionalization degree of 6%; orj. a chitosan-derivative nanoparticle, wherein the nanoparticlecomprises 24mer chitosan coupled with arginine at a finalfunctionalization degree of 52% and gluconic acid at a finalfunctionalization degree of 8%.

Polyplexes

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 110kDa, more preferably less than 65 kDa, more preferably less than 50 kDa,more preferably less than 40 kDa, and most preferably less than 30 kDabefore functionalization. In some embodiments, polyplexes of thecompositions comprise chitosan having an average molecular weight ofless than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDabefore functionalization.

In a preferred embodiment, the polyplexes comprise chitosan moleculeshaving on average less than 680 glucosamine monomer units, morepreferably less than 400 glucosamine monomer units, more preferably lessthan 310 glucosamine monomer units, more preferably less than 250glucosamine monomer units, and most preferably less than 190 glucosaminemonomer units. In some embodiments, the polyplexes comprise chitosanmolecules having on average less than 95 glucosamine monomer units, lessthan 65 glucosamine monomer units, less than 45 glucosamine monomerunits, or less than 35 glucosamine monomer units.

In a preferred embodiment, the subject polyplexes have amine tophosphate (N/P) ratio of 2 to 100, e.g., 2 to 50, e.g., 2 to 40, e.g., 2to 30, e.g., 2 to 20, e.g., 2 to 5. Preferably, the N/P ratio isinversely proportional to the molecular weight of the chitosan, i.e., asmaller molecular weight DD-chitosan requires a higher N/P ratio, andvice versa.

In a preferred embodiment, the subject polyplexes have an averagehydrodynamic diameter of less than 1000 nm, more preferably less than500 nm and most preferably less than 200 nm.

In one embodiment, the DD-chitosan nucleic acid polyplexes have anaverage zeta potential of at least 0 mV at an acidic pH, e.g., a pHbelow 7, most preferably a pH between about 4 to 6.

In one embodiment, the DD-chitosan nucleic acid polyplexes have anaverage zeta potential between +1 to +60 mV, more preferably +1 to +40mV, more preferably +1 to +30 mV at an acidic pH.

In a preferred embodiment, the polyplexes have a low net positive,neutral, or net negative charge at physiological pH and a pKa below 6.Such DD-chitosan nucleic acid polyplexes exhibit reduced cellulartoxicity and enhanced intracellular release of nucleic acid.

The DD-chitosan nucleic acid polyplexes of the composition arepreferably homogeneous in respect of polyplex size. Accordingly, in apreferred embodiment, the composition has a low average polydispersityindex (“PDI”). In an especially preferred embodiment, the DD-chitosannucleic acid polyplex dispersion has a PDI of less than 0.5, morepreferably less than 0.4, more preferably less than 0.3, and mostpreferably less than 0.25.

The polyplexes of the subject compositions are preferably substantiallysize stable in the composition. In a preferred embodiment, a compositionof the invention comprises polyplexes that increase in average diameterby less than 100%, more preferably less than 50%, and most preferablyless than 25%, at room temperature for 6 hours, more preferably 12hours, more preferably 24 hours, and most preferably 48 hours. In aparticularly preferred embodiment, a composition of the inventioncomprises polyplexes that increase in average diameter by less than 25%at room temperature for at least 24 hours or at least 48 hours.

The polyplexes of the subject compositions are preferably substantiallysize stable under cooled conditions. In a preferred embodiment, acomposition of the invention comprises polyplexes that increase inaverage diameter by less than 100%, more preferably less than 50%, andmost preferably less than 25%, at 2-8 degrees Celsius for 6 hours, morepreferably 12 hours, more preferably 24 hours, and most preferably 48hours.

The polyplexes of the subject compositions are preferably substantiallysize stable under freeze-thaw conditions. In a preferred embodiment, acomposition of the invention comprises polyplexes that increase inaverage diameter by less than 100%, more preferably less than 50%, andmost preferably less than 25% at room temperature for 6 hours, morepreferably 12 hours, more preferably 24 hours, and most preferably 48hours following thaw from frozen at −20 to −80 degrees Celsius.

In a preferred embodiment, the composition has a nucleic acidconcentration greater than 0.5 mg/ml, and is substantially free ofprecipitated polyplex. More preferably, the composition has a nucleicacid concentration of at least 0.6 mg/ml, more preferably at least 0.75mg/ml, more preferably at least 1.0 mg/ml, more preferably at least 1.2mg/ml, and most preferably at least 1.5 mg/ml, and is substantially freeof precipitated polyplex. The compositions are hydrated. In a preferredembodiment, the composition is substantially free of uncomplexed nucleicacid.

In a preferred embodiment, the DD-chitosan nucleic acid polyplexcomposition is isotonic. Achieving isotonicity, while maintainingpolyplex stability, is highly desirable in formulating pharmaceuticalcompositions, and these preferred compositions are well suited topharmaceutical formulation and therapeutic applications.

Generally, compositions comprising the DD-chitosan nucleic acidpolyplexes are used to contact a target cell. Such contact generallyresults in delivery of the nucleic acid for expression by the targetedcell. Compositions suitable for the DD-chitosan nucleic acid polyplexesdescribed herein are well known in the art, and are generally describedbelow.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 50kDa before functionalization, on average less than 310 glucosaminemonomer units, an N/P ratio of 2 to 20, an average hydrodynamic diameterof less than 500 nm, an average zeta potential of at least 0 mV at anacidic pH, a PDI of less than 0.5, a nucleic acid concentration greaterthan 0.5 mg/ml, are substantially free of precipitated polyplex, and thepolyplexes are size stable in that they increase in average diameter byless than 100% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 65kDa before functionalization, on average less than 400 glucosaminemonomer units, an N/P ratio of 2 to 20, an average hydrodynamic diameterof less than 500 nm, an average zeta potential of +1 to +40 mV at anacidic pH, a PDI of less than 0.4, a nucleic acid concentration greaterthan 0.5 mg/ml, are substantially free of precipitated polyplex, and thepolyplexes are size stable in that they increase in average diameter byless than 50% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 40kDa before functionalization, on average less than 250 glucosaminemonomer units, an N/P ratio of 2 to 20, an average hydrodynamic diameterof less than 500 nm, an average zeta potential of +1 to +30 mV at anacidic pH, a PDI of less than 0.3, a nucleic acid concentration greaterthan 0.5 mg/ml, are substantially free of precipitated polyplex, and thepolyplexes are size stable in that they increase in average diameter byless than 50% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 30kDa before functionalization, on average less than 190 glucosaminemonomer units, an N/P ratio of 2 to 5, an average hydrodynamic diameterof less than 500 nm, an average zeta potential of +1 to +30 mV at anacidic pH, a PDI of less than 0.25, a nucleic acid concentration greaterthan 0.5 mg/ml, are substantially free of precipitated polyplex, and thepolyplexes are size stable in that they increase in average diameter byless than 25% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 30kDa before functionalization, on average less than 190 glucosaminemonomer units, an N/P ratio of 2 to 5, an average hydrodynamic diameterof less than 500 nm, an average zeta potential of +1 to +30 mV at anacidic pH, a PDI of less than 0.25, a nucleic acid concentration greaterthan 0.75 mg/ml, are substantially free of precipitated polyplex, andthe polyplexes are size stable in that they increase in average diameterby less than 25% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

In a preferred embodiment, the polyplexes of the compositions comprisechitosan molecules having an average molecular weight of less than 15kDa before functionalization, on average less than 95 glucosaminemonomer units, an N/P ratio of 2 to 5, an average hydrodynamic diameterof less than 200 nm, an average zeta potential of +1 to +30 mV at anacidic pH, a PDI of less than 0.25, a nucleic acid concentration greaterthan 1.0 mg/ml, are substantially free of precipitated polyplex, and thepolyplexes are size stable in that they increase in average diameter byless than 50% at room temperature for at least 6 hours, at least 12hours, at least 24 hours, or at least 48 hours. In a preferredembodiment, the DD-chitosan nucleic acid polyplex composition isisotonic and are substantially size stable under freeze-thaw conditions.

Powdered Formulations

The DD-chitosan nucleic acid polyplex compositions of the inventioninclude powders. In a preferred embodiment, the invention provides a drypowder DD-chitosan nucleic acid polyplex composition. In a preferredembodiment, the dry powder DD-chitosan nucleic acid polyplex compositionis produced through the dehydration of a chitosan-nucleic acid polyplexdispersion of the invention.

Pharmaceutical Formulations

The present invention also provides “pharmaceutically acceptable” or“physiologically acceptable” formulations comprising DD-chitosan nucleicacid polyplex compositions of the invention. Such formulations can beadministered in vivo to a subject in order to practice treatmentmethods.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

Excipients can include a salt, an isotonic agent, a serum protein, abuffer or other pH-controlling agent, an anti-oxidant, a thickener, anuncharged polymer, a preservative or a cryoprotectant. Excipients usedin compositions of the invention may further include an isotonic agentand a buffer or other pH-controlling agent. These excipients may beadded for the attainment of preferred ranges of pH (about 6.0-8.0) andosmolarity (about 50-400 mmol/L). Examples of suitable buffers areacetate, borate, carbonate, citrate, phosphate and sulfonated organicmolecule buffer. Such buffers may be present in a composition inconcentrations from 0.01 to 1.0% (w/v). An isotonic agent may beselected from any of those known in the art, e.g. mannitol, dextrose,glucose and sodium chloride, or other electrolytes. Preferably, theisotonic agent is glucose or sodium chloride. The isotonic agents may beused in amounts that impart to the composition the same or a similarosmotic pressure as that of the biological environment into which it isintroduced. The concentration of isotonic agent in the composition willdepend upon the nature of the particular isotonic agent used and mayrange from about 0.1 to 10%. When glucose is used, it is preferably usedin a concentration of from 1 to 5% w/v, more particularly 5% w/v. Whenthe isotonic agent is sodium chloride, it is preferably employed inamounts of up to 1% w/v, in particular 0.9% w/v. The compositions of theinvention may further contain a preservative. Examples preservatives arepolyhexamethylene-biguanidine, benzalkonium chloride, stabilizedoxychloro complexes (such as those known as Purite®), phenylmercuricacetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol,parabens, and thimerosal. Typically, such preservatives are present atconcentrations from about 0.001 to 1.0%. Furthermore, the compositionsof the invention may also contain a cryopreservative agent. Preferredcryopreservatives are glucose, sucrose, mannitol, lactose, trehalose,sorbitol, colloidal silicon dioxide, dextran of molecular weightpreferable below 100,000 g/mol, glycerol, and polyethylene glycols ofmolecular weights below 100,000 g/mol or mixtures thereof. Mostpreferred are glucose, trehalose and polyethylene glycol. Typically,such cryopreservatives are present at concentrations from about 0.01 to10%.

A pharmaceutical formulation can be formulated to be compatible with itsintended route of administration. For example, for oral administration,a composition can be incorporated with excipients and used in the formof tablets, troches, capsules, e.g., gelatin capsules, or coatings,e.g., enteric coatings (Eudragit® or Sureteric®). Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included inoral formulations. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or other stearates; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint, methyl salicylate, or flavoring.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. For example, a time delay material such as glycerylmonostearate or glyceryl stearate alone, or in combination with a wax,may be employed.

Suppositories and other rectally administrable formulations (e.g., thoseadministrable by enema) are also contemplated. Further regarding rectaldelivery, see, for example, Song et al., Mucosal drug delivery:membranes, methodologies, and applications, Crit. Rev. Ther. Drug.Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein andpeptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. CarrierSyst., 8:331-394, 1991.

Additional pharmaceutical formulations appropriate for administrationare known in the art and are applicable in the methods and compositionsof the invention (see, e.g., Remington's Pharmaceutical Sciences (1990)18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, N.J.; and PharmaceuticalPrinciples of Solid Dosage Forms, Technonic Publishing Co., Inc.,Lancaster, Pa., (1993)).

Administration

In one embodiment, the use of DD-chitosan in DD-chitosan nucleic acidpolyplexes provides for prolonged stability of polyplexes atphysiological pH. This provides for effective systemic administration,as well as other modes of administration.

Any of a number of administration routes are possible and the choice ofa particular route will in part depend on the target tissue. Syringes,endoscopes, cannulas, intubation tubes, catheters and other articles maybe used for administration.

The doses or “effective amount” for treating a subject are preferablysufficient to ameliorate one, several or all of the symptoms of thecondition, to a measurable or detectable extent, although preventing orinhibiting a progression or worsening of the disorder or condition, or asymptom, is a satisfactory outcome. Thus, in the case of a condition ordisorder treatable by expressing a therapeutic nucleic acid in targettissue, the amount of therapeutic RNA or therapeutic protein produced toameliorate a condition treatable by a method of the invention willdepend on the condition and the desired outcome and can be readilyascertained by the skilled artisan. Appropriate amounts will depend uponthe condition treated, the therapeutic effect desired, as well as theindividual subject (e.g., the bioavailability within the subject,gender, age, etc.). The effective amount can be ascertained by measuringrelevant physiological effects.

Veterinary applications are also contemplated by the present invention.Accordingly, in one embodiment, the invention provides methods oftreating non-human mammals, which involve administering a chitosan-basednanoparticle of the invention to a non-human mammal in need oftreatment.

Parenteral Administration

The compounds of the invention may be administered directly into theblood stream, into muscle, or into an internal organ. Suitable means forparenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular and subcutaneous. Suitabledevices for parenteral administration include needle (includingmicroneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents,but, for some applications, they may be more suitably formulated as asterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilisation, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The solubility of compounds used in the preparation of parenteralsolutions may be increased by the use of appropriate formulationtechniques, such as the incorporation of solubility-enhancing agents.

Formulations for parenteral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease. Thus compounds of the invention may be formulated as a solid,semi-solid, or thixotropic liquid for administration as an implanteddepot providing modified release of the active compound.

Oral Administration

The subject compositions may be administered orally. Oral administrationmay involve swallowing, so that the compound enters the gastrointestinaltract. Compositions of the invention may also be administered directlyto the gastrointestinal tract.

Formulations suitable for oral administration include solid formulationssuch as tablets, capsules, coated capsules containing particulates orcoated particulates, liquids, or powders, lozenges (includingliquid-filled), chews, multi- and nano-particulates, gels, films,ovules, and sprays.

Liquid formulations include suspensions, solutions, syrups and elixirs.Liquid formulations may be prepared by the reconstitution of a solid.

Tablet dosage forms generally contain a disintegrant. Examples ofdisintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, croscarmellose sodium,crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystallinecellulose, lower alkyl-substituted hydroxypropyl cellulose, starch,pregelatinised starch and sodium alginate. Generally, the disintegrantwill comprise from 1 weight % to 25 weight %, preferably from 5 weight %to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tabletformulation. Suitable binders include microcrystalline cellulose,gelatin, sugars, polyethylene glycol, natural and synthetic gums,polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose andhydroxypropyl methylcellulose. Tablets may also contain diluents, suchas lactose (monohydrate, spray-dried monohydrate, anhydrous and thelike), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystallinecellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such assodium lauryl sulfate and polysorbate 80, and glidants such as silicondioxide and talc. When present, surface active agents may comprise from0.2 weight % to 5 weight % of the tablet, and glidants may comprise from0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate,calcium stearate, zinc stearate, sodium stearyl fumarate, and mixturesof magnesium stearate with sodium lauryl sulphate. Lubricants generallycomprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight %to 3 weight % of the tablet.

Other possible ingredients include anti-oxidants, colorants, flavoringagents, preservatives and taste-masking agents.

Tablet blends may be compressed directly or by roller to form tablets.Tablet blends or portions of blends may alternatively be wet-, dry-, ormelt-granulated, melt congealed, or extruded before tabletting. Thefinal formulation may comprise one or more layers and may be coated oruncoated; it may even be encapsulated.

The formulation of tablets is discussed in Pharmaceutical Dosage Forms:Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, NewYork, 1980).

Consumable oral films for human or veterinary use are typically pliablewater-soluble or water-swellable thin film dosage forms which may berapidly dissolving or mucoadhesive and typically comprise a film-formingpolymer, a binder, a solvent, a humectant, a plasticiser, a stabiliseror emulsifier, a viscosity-modifying agent and a solvent. Somecomponents of the formulation may perform more than one function.

Also included in the invention are multiparticulate beads comprising acomposition of the invention.

Other possible ingredients include anti-oxidants, colorants, flavouringsand flavour enhancers, preservatives, salivary stimulating agents,cooling agents, co-solvents (including oils), emollients, bulkingagents, anti-foaming agents, surfactants and taste-masking agents.

Films in accordance with the invention are typically prepared byevaporative drying of thin aqueous films coated onto a peelable backingsupport or paper. This may be done in a drying oven or tunnel, typicallya combined coater dryer, or by freeze-drying or vacuuming.

Solid formulations for oral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

Other suitable release technologies such as high energy dispersions andosmotic and coated particles are known.

Topical Administration

The compounds of the invention may also be administered topically to theskin or mucosa, that is, dermally or transdermally. Typical formulationsfor this purpose include gels, hydrogels, lotions, solutions, creams,ointments, dusting powders, dressings, foams, films, skin patches,wafers, implants, sponges, fibres, bandages and microemulsions.

Other means of topical administration include delivery byelectroporation, iontophoresis, phonophoresis, sonophoresis andmicroneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

Formulations for topical administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

Inhaled/Intranasal Administration

The compounds of the invention can also be administered intranasally orby inhalation, typically in the form of a dry powder (either alone, as amixture, for example, in a dry blend with lactose, or as a mixedcomponent particle) from a dry powder inhaler or as an aerosol sprayfrom a pressurized container, pump, spray, atomiser, or nebuliser, withor without the use of a suitable propellant.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the compound of theinvention, a suitable powder base such as lactose or starch and aperformance modifier such as I-leucine, mannitol, or magnesium stearate.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled-, targeted andprogrammed release.

Rectal/Intravaginal Administration

The compounds of the invention may be administered rectally orvaginally, for example, in the form of a suppository, pessary, or enema.Cocoa butter is a traditional suppository base, but various alternativesmay be used as appropriate.

Formulations for rectal/vaginal administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

Ocular/Aural Administration

The compounds of the invention may also be administered directly to theeye or ear, typically in the form of drops. Other formulations suitablefor ocular and aural administration include ointments, biodegradable(e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g.silicone) implants, wafers, lenses and particulate systems. Formulationsmay also be delivered by iontophoresis.

Formulations for ocular/aural administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted, or programmedrelease.

Methods of Use

In one embodiment, DD-chitosan nucleic acid polyplex compositions of theinvention may be used for therapeutic treatment. Such compositions aresometimes referred to herein as therapeutic compositions.

Therapeutic proteins of the invention, as discussed below, are producedby polyplexes of the invention comprising therapeutic nucleic acids. Useof the subject proteins as described below refers to use of the subjectpolyplexes to affect such protein use.

Therapeutic proteins contemplated for use in the invention have a widevariety of activities and find use in the treatment of a wide variety ofdisorders. The following description of therapeutic protein activities,and indications treatable with therapeutic proteins of the invention, isexemplary and not intended to be exhaustive. The term “subject” refersto an animal, with mammals being preferred, and humans being especiallypreferred.

A partial list of therapeutic proteins and target diseases is shown inTable 4.

TABLE 4 LEAD TARGET THERAPEUTIC COMPOUNDS DISEASE FUNCTION EFFECTInsulin Diabetes Insulin replacement Improve glucose tolerance.Delay/prevent diabetes. Glucagon Diabetes Reduce endogenous Improveglucose antagonists glucose production tolerance GLP-1 DiabetesStimulate growth of Improve glucose Obesity ß-cells, improve tolerance.insulin sensitivity, Induce weight loss suppress appetite Leptin ObesityAppetite suppression Induce weight loss. Diabetes and improvement ofImprove glucose insulin sensitivity tolerance CCK Obesity Appetitesuppression Induce weight loss Growth Hormone GH deficiencies, GHreplacement Improve growth (GH) wasting and anti- aging Clotting factorsHemophilia Clotting factors Improve clotting replacement timeTherapeutic Infections Pathogen Prevent infections antibodies and Cancerneutralization or or transplant antibody immune modulations rejectionsfragments/portions Inflammation Gastrointestinal Immune modulationPrevent inhibitors, e.g., IL- organ inflammation; inflammation in 10,TGF-β, TNFα e.g., inflammatory Gastrointestinal antagonists, IL-17 boweldisease (IBD) organ antagonists

In another embodiment, therapeutic compositions of the inventioncomprise therapeutic nucleic acids that do not encode therapeuticproteins, e.g., therapeutic RNAs. For example, by selecting therapeuticRNAs that target genes involved in mechanisms of disease and/orundesirable cellular or physiological conditions, the subjectcompositions may be used in the treatment of a wide array of diseasesand conditions. The subject compositions are of such character that thetherapeutic RNAs used are not limited in respect of the scope of targetselection. Accordingly, the subject compositions find use in any diseaseor condition involving a suitable target.

Preferred tissues, diseases, and conditions include the following, whichare exemplary and in no way limiting:

Target Organ Target Disease Gastrointestinal (GI) organs DiabetesObesity Inflammatory bowel disease Irritable bowel syndrome GI infectionPeptic ulcers Gastroesophageal reflux Gastroparesis HemorrhoidsMalabsorption of nutrients GI cancers (colorectal, pancreatic, stomach,esophageal, bile duct, gall bladder cancers) PancreatitisHemochromatosis Celiac disease Food allergies Immune tolerance inductionHormone deficiency Eye Macular degeneration Age-related maculardegeneration Uveitis Retinitis pigmentosa Iritis Scleritis GlaucomaKeratititis Retinopathy Eye infection (e.g. keratomycosis) Uterus,vagina, ovary and Cancers cervix Infections Endometriosis CervicitisUrologic pain Polyps Fibroids Endometrial hyperplasia Bladder andurinary tract Urinary incontinence Bladder and urinary tract infectionOveractive bladder Erectile dysfunction Diabetic neuropathy KidneyDiabetic nephropathy Membranous nephropathy Hypertension Renal cancerHypertension Polycystic kidney disease Glomerulonephritis LiverDyslipidemia/hypercholesterolemia Diabetes Metabolic syndrome HepatomaHepatitis A/B/C Hemochromatosis Cirrhosis Steatohepatitis Glycogenstorage diseases Skin Psoriasis Acne Rosacea Granulomatous dermatitisAnti-wrinkle Depigmentation Lung/Respiratory organs Lung cancer Chronicobstructive pulmonary disease Respiratory tract infection Cysticfibrosis Pulmonary vascular diseases Myasthenia gravis Fibrosis AsthmaBrain Huntington's disease Alzheimer disease Parkinson's disease Braincancer Obesity Neurological disorders Blood cells Cancers Infectiousdisease Autoimmune disease Muscle Metabolic syndrome AtherosclerosisDiabetes Sarcoma Inflammation (e.g. polymyositis) Glycogen storagediseases Myopathy Heart Myocardial infarction Atherosclerosis AnginaCardiomyopathy Ischemia Hypertensive heart diseases Thrombosis AneurysmAdipose Diabetes Obesity Metabolic syndrome Atherosclerosis Dyslipidemia

Hyperglycemia and Body Mass

Therapeutic proteins include insulin and insulin analogs. Diabetesmellitus is a debilitating metabolic disease caused by absent (type 1)or insufficient (type 2) insulin production from pancreatic 3-cells(Unger, R. H. et al., Williams Textbook of Endocrinology Saunders,Philadelphia (1998)). Beta-cells are specialized endocrine cells thatmanufacture and store insulin for release following a meal (Rhodes, et.al. J. Cell Biol. 105:145(1987)) and insulin is a hormone thatfacilitates the transfer of glucose from the blood into tissues where itis needed. Patients with diabetes must frequently monitor blood glucoselevels and many require multiple daily insulin injections to survive.However, such patients rarely attain ideal glucose levels by insulininjection (Turner, R. C. et al. JAMA 281:2005(1999)). Furthermore,prolonged elevation of insulin levels can result in detrimental sideeffects such as hypoglycemic shock and desensitization of the body'sresponse to insulin. Consequently, diabetic patients still developlong-term complications, such as cardiovascular diseases, kidneydisease, blindness, nerve damage and wound healing disorders (UKProspective Diabetes Study (UKPDS) Group, Lancet 352, 837 (1998)).

Disorders treatable by a method of the invention include a hyperglycemiccondition, such as insulin-dependent (type 1) or -independent (type 2)diabetes, as well as physiological conditions or disorders associatedwith or that result from the hyperglycemic condition. Thus,hyperglycemic conditions treatable by a method of the invention alsoinclude a histopathological change associated with chronic or acutehyperglycemia (e.g., diabetes). Particular examples include degenerationof pancreas (β-cell destruction), kidney tubule calcification,degeneration of liver, eye damage (diabetic retinopathy), diabetic foot,ulcerations in mucosa such as mouth and gums, excess bleeding, delayedblood coagulation or wound healing and increased risk of coronary heartdisease, stroke, peripheral vascular disease, dyslipidemia, hypertensionand obesity.

The subject compositions are useful for decreasing glucose, improvingglucose tolerance, treating a hyperglycemic condition (e.g., diabetes)or for treating a physiological disorders associated with or resultingfrom a hyperglycemic condition. Such disorders include, for example,diabetic neuropathy (autonomic), nephropathy (kidney damage), skininfections and other cutaneous disorders, slow or delayed healing ofinjuries or wounds (e.g., that lead to diabetic carbuncles), eye damage(retinopathy, cataracts) which can lead to blindness, diabetic foot andaccelerated periodontitis. Such disorders also include increased risk ofdeveloping coronary heart disease, stroke, peripheral vascular disease,dyslipidemia, hypertension and obesity.

As used herein, the term “hyperglycemic” or “hyperglycemia,” when usedin reference to a condition of a subject, means a transient or chronicabnormally high level of glucose present in the blood of a subject. Thecondition can be caused by a delay in glucose metabolism or absorptionsuch that the subject exhibits glucose intolerance or a state ofelevated glucose not typically found in normal subjects (e.g., inglucose-intolerant subdiabetic subjects at risk of developing diabetes,or in diabetic subjects). Fasting plasma glucose (FPG) levels fornormoglycemia are less than about 110 mg/dl, for impaired glucosemetabolism, between about 110 and 126 mg/dl, and for diabetics greaterthan about 126 mg/dl.

Disorders treatable by producing a protein in a gut mucosal tissue alsoinclude obesity or an undesirable body mass. Leptin, cholecystokinin,PYY and GLP-1 decrease hunger, increase energy expenditure, induceweight loss or provide normal glucose homeostasis. Thus, in variousembodiments, a method of the invention for treating obesity or anundesirable body mass, or hyperglycemia, involves the use of atherapeutic nucleic acid encoding leptin, cholecystokinin, PYY or GLP-1.In another embodiment, a therapeutic RNA targeting ghrelin is used.Ghrelin increases appetite and hunger. Thus, in various embodiments, amethod of the invention for treating obesity or an undesirable bodymass, or hyperglycemia, involves the use of a therapeutic RNA targetingghrelin to decrease the expression thereof. Disorders treatable alsoinclude those typically associated with obesity, for example, abnormallyelevated serum/plasma LDL, VLDL, triglycerides, cholesterol, plaqueformation leading to narrowing or blockage of blood vessels, increasedrisk of hypertension/stroke, coronary heart disease, etc.

As used herein, the term “obese” or “obesity” refers to a subject havingat least a 30% increase in body mass in comparison to an age and gendermatched normal subject. “Undesirable body mass” refers to subjectshaving 1%-29% greater body mass than a matched normal subject as well assubjects that are normal with respect to body mass but who wish todecrease or prevent an increase in their body mass.

In one embodiment, a therapeutic protein of the invention is a glucagonantagonist. Glucagon is a peptide hormone produced by β-cells inpancreatic islets and is a major regulator of glucose metabolism (UngerR. H. & Orci L. N. Eng. J. Med. 304:1518(1981); Unger R. H. Diabetes25:136 (1976)). As with insulin, blood glucose concentration mediatesglucagon secretion. However, in contrast to insulin glucagon is secretedin response to a decrease in blood glucose. Therefore, circulatingconcentrations of glucagon are highest during periods of fast and lowestduring a meal. Glucagon levels increase to curtail insulin frompromoting glucose storage and stimulate liver to release glucose intothe blood. A specific example of a glucagon antagonist is [des-His1,des-Phe6, Glu9]glucagon-NH2. In streptozotocin diabetic rats, bloodglucose levels were lowered by 37% within 15 min of an intravenous bolus(0.75 μg/g body weight) of this glucagon antagonist (Van Tine B. A. et.al. Endocrinology 137:3316 (1996)). In another embodiment, the inventionprovides a method for treating diabetes or hyperglycemia, comprising theuse of a therapeutic RNA to decrease the levels of glucagon productionfrom the pancreas.

In another embodiment, a therapeutic protein of the invention useful fortreating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is a glucagon-like peptide-1 (GLP-1). GLP-1 is a hormonereleased from L-cells in the intestine during a meal which stimulatespancreatic β-cells to increase insulin secretion. GLP-1 has additionalactivities that make it an attractive therapeutic agent for treatingobesity and diabetes. For example, GLP-1 reduces gastric emptying,suppresses appetite, reduces glucagon concentration, increases β-cellmass, stimulates insulin biosynthesis and secretion in aglucose-dependent fashion, and likely increases tissue sensitivity toinsulin (Kieffer T. J., Habener J. F. Endocrin. Rev. 20:876 (2000)).Therefore, regulated release of GLP-1 in the gut to coincide with a mealcan provide therapeutic benefit for a hyperglycemic condition or anundesirable body mass. GLP-1 analogs that are resistant to dipeptidylpeptidase IV (DPP IV) provide longer duration of action and improvedtherapeutic value. Thus, GLP-1 analogs are preferred therapeuticpolypeptides. In another embodiment, the invention provides a method fortreating diabetes or hyperglycemia, comprising the use of a therapeuticRNA to decrease the levels of DPP IV.

In another embodiment, a therapeutic protein of the invention useful fortreating a hyperglycemic condition is an antagonist to the hormoneresistin. Resistin is an adipocyte-derived factor for which expressionis elevated in diet-induced and genetic forms of obesity. Neutralizationof circulating resistin improves blood glucose and insulin action inobese mice. Conversely, administration of resistin in normal miceimpairs glucose tolerance and insulin action (Steppan C M et. al. Nature409:307 (2001)). Production of a protein that antagonizes the biologicaleffects of resistin in gut can therefore provide an effective therapyfor obesity-linked insulin resistance and hyperglycemic conditions. Inanother embodiment, the invention provides a method for treatingdiabetes or hyperglycemia, comprising the use of a therapeutic RNA todecrease the levels of resistin expression in adipose tissue.

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is leptin. Leptin, although produced primarily by fat cells, isalso produced in smaller amounts in a meal-dependent fashion in thestomach. Leptin relays information about fat cell metabolism and bodyweight to the appetite centers in the brain where it signals reducedfood intake (promotes satiety) and increases the body's energyexpenditure.

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is the C-terminal globular head domain of adipocytecomplement-related protein (Acrp30). Acrp30 is a protein produced bydifferentiated adipocytes. Administration of a proteolytic cleavageproduct of Acrp30 consisting of the globular head domain to mice leadsto significant weight loss (Fruebis J. et al. Proc. Natl Acad. Sci USA98:2005 (2001)).

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptidesecreted from the intestine in response to particular nutrients in thegut. CCK release is proportional to the quantity of food consumed and isbelieved to signal the brain to terminate a meal (Schwartz M. W. et. al.Nature 404:661-71(2000)). Consequently, elevated CCK can reduce mealsize and promote weight loss or weight stabilization (i.e., prevent orinhibit increases in weight gain).

Regarding PYY, see for example le Roux et al., Proc Nutr Soc. 2005 May;64(2):213-6.

Immunological Disorders

In one embodiment, a therapeutic composition of the invention possessesimmunomodulatory activity. For example, a therapeutic polypeptide of thepresent invention may be useful in treating deficiencies or disorders ofthe immune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through the process of hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune deficiencies or disorders may be genetic, somatic, such ascancer or some autoimmune disorders, acquired (e.g. by chemotherapy ortoxins), or infectious.

A therapeutic composition of the present invention may be useful intreating deficiencies or disorders of hematopoietic cells. For example,a therapeutic polypeptide of the present invention could be used toincrease differentiation or proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat thosedisorders associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, DiGeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

A therapeutic composition of the present invention may also be useful intreating autoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Accordingly, the administration of atherapeutic composition of the present invention that inhibits an immuneresponse, particularly the proliferation, differentiation, or chemotaxisof T-cells, may be an effective therapy in preventing autoimmunedisorders.

Examples of autoimmune disorders that can be treated by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin-dependent diabetes mellitus, Crohn'sdisease, ulcerative colitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a therapeutic composition of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A therapeutic composition of the present invention may also be used totreat and/or prevent organ rejection or graft-versus-host disease(GVHD). Organ rejection occurs by host immune cell destruction of thetransplanted tissue through an immune response. Similarly, an immuneresponse is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. The administrationof a therapeutic composition of the present invention that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, a therapeutic composition of the present invention may alsobe used to modulate inflammation. For example, the therapeuticpolypeptide may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat inflammatory conditions, both chronic and acute conditions,including inflammation associated with infection (e.g. septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,pancreatitis, complement-mediated hyperacute rejection, nephritis,cytokine or chemokine induced lung injury, inflammatory bowel disease(IBD), Crohn's disease, or resulting from over production of cytokines(e.g. TNF or IL-1.) In one embodiment, a therapeutic RNA targetedagainst TNFα is used in the subject compositions to treat inflammation.In another preferred embodiment, a therapeutic RNA targeted against IL-1is used in the subject compositions to treat inflammation. siRNAtherapeutic RNAs are especially preferred. Inflammatory disorders ofinterest for treatment in the present invention include, but are notlimited to, chronic obstructive pulmonary disorder (COPD), interstitialcystitis, and inflammatory bowel disease.

Clotting Disorders

In some embodiments, a therapeutic composition of the present inventionmay also be used to modulate hemostatic (the stopping of bleeding) orthrombolytic activity (clot formation). For example, by increasinghemostatic or thrombolytic activity, a therapeutic composition of thepresent invention could be used to treat blood coagulation disorders(e.g. afibrinogenemia, factor deficiencies), blood platelet disorders(e.g. thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, a therapeutic composition of the presentinvention that can decrease hemostatic or thrombolytic activity could beused to inhibit or dissolve clotting. These therapeutic compositionscould be important in the treatment of heart attacks (infarction),strokes, or scarring. In one embodiment, a therapeutic polypeptide ofthe invention is a clotting factor, useful for the treatment ofhemophilia or other coagulation/clotting disorders (e.g., Factor VIII,IX or X)

Hyperproliferative Disorders

In one embodiment, a therapeutic composition of the invention is capableof modulating cell proliferation. Such a therapeutic polypeptide can beused to treat hyperproliferative disorders, including neoplasms.

Examples of hyperproliferative disorders that can be treated by atherapeutic composition of the present invention include, but are notlimited to neoplasms located in the: abdomen, bone, breast, digestivesystem, liver, pancreas, peritoneum, endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, headand neck, nervous (central and peripheral), lymphatic system, pelvic,skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated by atherapeutic composition of the present invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

Delivery to the circulatory system provides for access of therapeuticprotein to a wide variety of tissues. Alternatively, a therapeuticcomposition of the present invention may stimulate the proliferation ofother cells that can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as with a chemotherapeutic agent.

Infectious Disease

In one embodiment, a therapeutic composition of the present inventioncan be used to treat infectious disease. For example, by increasing theimmune response, particularly increasing the proliferation anddifferentiation of B and/or T cells, infectious diseases may be treated.The immune response may be increased by either enhancing an existingimmune response, or by initiating a new immune response. Alternatively,the therapeutic composition of the present invention may also directlyinhibit the infectious agent, without necessarily eliciting an immuneresponse.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated by a therapeutic composition of the presentinvention. Examples of viruses, include, but are not limited to thefollowing DNA and RNA viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis),Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),Mononegavirus (e.g. Paramyxoviridae, Morbillivirus, Rhabdoviridae),Orthomyxoviridae (e.g. Influenza), Papovaviridae, Parvoviridae,Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae(e.g. Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g. Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiolitis, encephalitis, eye infections (e.g.conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g.AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever,Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia,Rubella, sexually transmitted diseases, skin diseases (e.g. Kaposi's,warts), and viremia. A therapeutic composition of the present inventioncan be used to treat any of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated by a therapeutic composition of the presentinvention include, but are not limited to, the following Gram-Negativeand Gram-positive bacterial families and fungi: Actinomycetales (e.g.Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae(e.g. Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g. Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections(e.g. Actinobacillus, Heamophilus, Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. Thesebacterial or fungal families can cause the following diseases orsymptoms, including, but not limited to: bacteremia, endocarditis, eyeinfections (conjunctivitis, tuberculosis, uveitis), gingivitis,opportunistic infections (e.g. AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis,Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism,gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexuallytransmitted diseases, skin diseases (e.g. cellulitis, dermatocycoses),toxemia, urinary tract infections, wound infections. A therapeuticcomposition of the present invention can be used to treat any of thesesymptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can betreated by a therapeutic composition of the present invention include,but are not limited to, the following families: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g. dysentery,giardiasis), liver disease, lung disease, opportunistic infections (e.g.AIDS related), Malaria, pregnancy complications, and toxoplasmosis. Atherapeutic composition of the present invention can be used to treatany of these symptoms or diseases.

Regeneration

A therapeutic composition of the present invention can be used todifferentiate, proliferate, and attract cells, fostering theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, bums, incisions,or ulcers), age, disease (e.g. osteoporosis, osteoarthritis, periodontaldisease, liver failure), surgery, including cosmetic plastic surgery,fibrosis, reperfusion injury, or systemic cytokine damage.

Therapeutic compositions of the invention may promote the regenerationof a variety of tissues, including but not limited to organs (e.g.pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,skeletal or cardiac), vascular (including vascular endothelium),nervous, hematopoietic, and skeletal (bone, cartilage, tendon, andligament) tissue. Preferably, regeneration incurs a small amount ofscarring, or occurs without scarring. Regeneration also may includeangiogenesis.

Moreover, a therapeutic composition of the present invention mayincrease regeneration of tissues difficult to heal. For example,increased tendon/ligament regeneration would quicken recovery time afterdamage. A therapeutic composition of the present invention could also beused prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using atherapeutic composition of the present invention to proliferate anddifferentiate nerve cells. Diseases that could be treated using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic disorders (e.g. spinal corddisorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g. resulting from chemotherapy or other medicaltherapies), localized neuropathies, and central nervous system diseases(e.g. Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all betreated using therapeutic compositions of the present invention. Withrespect to CNS disorders, numerous means are known in the art forfacilitating therapeutic access to brain tissue, including methods fordisrupting the blood brain barrier, and methods of coupling therapeuticagents to moieties that provide for transport into the CNS. In oneembodiment, a therapeutic nucleic acid is engineered so as to encode afusion protein, which fusion protein comprises a transport moiety and atherapeutic protein. Alternatively, the subject compositions may bedelivered directly to the CNS.

Chemotaxis

In one embodiment, a therapeutic composition of the invention canmodulate chemotaxis. For example, in one embodiment, a therapeuticpolypeptide of the present invention possesses a chemotaxis activity. Achemotaxic molecule attracts or mobilizes cells (e.g. monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

For example, a therapeutic polypeptide of the present invention mayincrease chemotaxic activity of particular cells. These chemotaxicmolecules can then be used to treat inflammation, infection,hyperproliferative disorders, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat wounds andother trauma to tissues by attracting immune cells to the injuredlocation. Chemotaxic molecules of the present invention can also attractfibroblasts, which can be used to treat wounds.

It is also contemplated that a therapeutic composition of the presentinvention may inhibit chemotaxic activity. These therapeuticcompositions could also be used to treat disorders. Thus, a therapeuticcomposition of the present invention could be used as an inhibitor ofchemotaxis.

Especially preferred for use are protherapeutic proteins that areactivated in the vicinity of target tissues.

Additional therapeutic polypeptides contemplated for use include, butare not limited to, growth factors (e.g., growth hormone, insulin-likegrowth factor-1, platelet-derived growth factor, epidermal growthfactor, acidic and basic fibroblast growth factors, transforming growthfactor-β, etc.), to treat growth disorders or wasting syndromes; andantibodies (e.g., human or humanized), to provide passive immunizationor protection of a subject against foreign antigens or pathogens (e.g.,H. Pylori), or to provide treatment of cancer, arthritis orcardiovascular disease; cytokines, interferons (e.g., interferon (IFN),IFN-α2b and 2α, IFN-α N1, IFN-β11b, IFN-gamma), interleukins (e.g., IL-1to IL-10), tumor necrosis factor (TNF-α TNF-β), chemokines, granulocytemacrophage colony stimulating factor (GM-CSF), polypeptide hormones,antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral,and/or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase),gonadotrophins, chemotactins, lipid-binding proteins, filgastim(Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase,sarbramostim, tissue plasminogen activator (tPA), urokinase,streptokinase, phenylalanine ammonia lyase, brain-derived neurotrophicfactor (BDNF), nerve growth factor (NGF), thrombopoietin (TPO),superoxide dismutase (SOD), adenosine deamidase, catalase, calcitonin,endothelin, L-asparaginase pepsin, uricase, trypsin, chymotrypsin,elastase, carboxypeptidase, lactase, sucrase, intrinsic factor,parathyroid hormone (PTH)-like hormone, soluble CD4, and antibodiesand/or antigen-binding fragments (e.g, FAbs) thereof (e.g., orthocloneOKT-e (anti-CD3), GPIIb/IIa monoclonal antibody). Additionallycontemplated are therapeutic RNAs targeting nucleic acids encoding suchfactors.

Vaccine

In one embodiment, the invention provides methods for vaccinating apatient. The methods comprise administering a composition of theinvention capable of producing the desired epitope. In a preferredembodiment, the composition comprises a therapeutic nucleic acidconstruct capable of expressing a protein comprising the epitope.

Cosmetic Applications

In one embodiment, the invention provides DD-chitosan nucleic acidpolyplexes for cosmetic use. The subject cosmetics comprise DD-chitosannucleic acid polyplexes in a formulation suitable for cosmetic use.

EXAMPLES Example 1: Formation of Dually Derivatized Chitosan andFormation of DNA Polyplexes

Chitosan was dually derivatized (DD-chitosan) with arginine and gluconicacid, or with arginine and threonic acid, according to well-knownmethods. DD-chitosan was polyplexed with either a DNA vector encodingfor secreted alkaline phosphatase (SEAP) or luciferase siRNA.

Example 2: In Vitro Transfection with DNA Polyplex

In general, in vitro transfection of 293T cells with DD-chitosan nucleicacid polyplex formulations was performed in two steps: preparation ofcells followed by transfection.

Example 3: Maintenance of Cell Lines

The 293T cell line was courtesy of Dr. Timothy Kieffer's lab at theUniversity of British Columbia (Vancouver, Canada) and were prepared asfollows. Human kidney cells were transformed with the SV40 T-antigen;grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) containing10% fetal bovine serum (FBS) and penicillin/streptomycin; and maintainedbelow 80% confluency.

Example 4: Preparation of Cells for Transfection

Cells were prepared for transfection as follows. On the day beforetransfection, 293T cells were added to 6-well tissue culture plates(4.5×10⁵ cells/well) in 3 mL of complete media (high glucose DMEM+10%FBS+pen/strep). On the day of transfection, cell count was determinedfor two selected wells by washing cells 1× with phosphate bufferedsaline (PBS) trypsinizing cells with 0.5 mL of 0.05% trypsin, adding 0.5mL of complete media and counting 10 μL using a hemocytometer. If cellswere ˜50% confluent (˜7×10⁵ cells/well), then transfection proceeded.(If cells were too sparse or too confluent, then transfection did notproceed.)

Example 5: Transfection of Cells

Transfection was carried out as follows. First, media was removed fromeach well followed by addition of 1 mL Opti-mem (pH 7.4) to each well,swirling gently and then removal. (Six wells were washed at a time toprevent cells from dislodging.) Then another 1 mL of Opti-mem (pH 7.4)was added carefully to each well so as not to dislodge cells. Next,polyplex samples were added to each well (target of 2 μg DNA), swirledand incubated at 37° C. for 2 h. After incubation, the media was removedand replaced with 2 mL of complete media and re-incubated at 37° C. Atthe required time points, the supernatant was removed and stored at −20°C. for subsequent SEAP assay.

Example 6: SEAP Protein Assay

The SEAP assay was performed using the SEAP Chemiluminescent Assay kit.All reagents for the assay were equilibrated at 25° C. for 30 min beforeuse. Standards for the assay were prepared by dissolving placentalalkaline phosphatase to 1 mg/mL in 1× dilution buffer from the kitspiked with 0.1% bovine serum albumin and 50% glycerol and then dilutingby 10-fold serial dilutions with DMEM to 0.01 pg/uL. Standards andthawed samples were then diluted 1 in 4 with dilution buffer, heatinactivated at 65° C. for 30 min, incubated on ice for 2 min,centrifuged (16100×rcf for 2 min at RT) and the supernatants transferredto new tubes. After equilibrating at 25° C. for 5 min, 50 uL of thesamples and standards were added to each well of a Microlite-1 plate induplicate. Inactivation buffer (50 uL) was then added to each well andpipetted up and down gently to mix, without creating bubbles andincubated for 5 min. The substrate/enhancer reagent was prepared duringthe 5 min incubation at a ratio for 1:19 of substrate to enhancer. Thesubstrate/enhancer was then added to each well, incubated for 20 min andthen the plate was read in the luminometer (Lmax11384, MolecularDevices) with an integration time of 1 sec.

Example 7: Results

FIGS. 1-3, show the transfection efficiencies of chitosan derivatizedwith different % of arginine or gluconic acid, and chitosan duallyderivatized with both gluconic acid and arginine. FIGS. 1-3 show asynergistic effect when chitosan is dually derivatized with botharginine and gluconic acid. The synergistic effect may be seen whenchitosan is dually functionalized with arginine at a finalfunctionalization degree of 10% and gluconic acid at finalfunctionalization degrees from 3% and 10%, although the greatest effectwas seen at a gluconic acid final functionalization degree of about 3%(FIG. 1). The synergistic effect may also be seen when chitosan isdually functionalized with arginine at a final functionalization degreeof 52% and gluconic acid at final functionalization degrees ranging from3% to 8%, although the greatest effect was seen at a gluconic acid finalfunctionalization degree of about 8% (FIG. 2). In addition, chitosandually functionalized with arginine and gluconic acid at finalfunctionalization degrees of 26% and 6% demonstrated greatestsynergistic effect in transfection efficiency (FIG. 3). Finally,synergistic effect may also be seen when chitosan is dually derivatizedwith arginine and an alternative moiety such as threonic acid instead ofgluconic acid. The synergistic effect is observed when chitosan isdually functionalized with arginine at a final functionalization degreeof 29% and threonic acid at final functionalization degrees ranging from3% (FIG. 4).

Example 8

Chitosan is dually derivatized (DD-chitosan) with arginine and an HPselected from the group consisting of 2,3-dihydroxylpropanoic acid;2,3,4,5,6,7-hexahydroxylheptanal; 2,3,4,5,6-pentahydroxylhexanal;2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal, and assayedaccording to Examples 1-7. DD-chitosan is polyplexed with either a DNAvector encoding for secreted alkaline phosphatase (SEAP) or luciferasesiRNA.

All citations are expressly incorporated herein in their entirety byreference.

All patents and patent publications referred to herein are herebyincorporated by reference.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

1-19. (canceled)
 20. A method of delivering a nucleic acid to a subjectin need thereof comprising administering to the subject a of a duallyderivatized (DD)-chitosan nucleic acid polyplex, wherein said DDchitosan nucleic acid polyplex comprises a nucleic acid complexed with achitosan-derivative nanoparticle comprising chitosan functionalized witharginine (Arg) and an hydrophilic polyol (HP) of Formula VII:

wherein: R² is selected from: H and hydroxyl; R³ is selected from H andhydroxyl; and X is selected from: C₂-C₆ alkylene optionally substitutedwith one or more hydroxyl substituents; provided said hydrophilic polyolis not gluconic acid.
 21. The method of claim 20, wherein saidhydrophilic polyol of the chitosan-derivative nanoparticle has acarboxyl group.
 22. The method of claim 21, wherein said hydrophilicpolyol is threonic acid.
 23. The method of claim 20, wherein saidhydrophilic polyol of said chitosan-derivative nanoparticle is asaccharide selected from the group consisting of glyceraldehyde,threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose,altrose, mannose, gulose, idose, galactose, and talose.
 24. The methodof claim 20, wherein said hydrophilic polyol of said chitosan-derivativenanoparticle is selected from the group consisting of2,3-dihydroxylpropanoic acid; 2,3,4,5,6,7-hexahydroxylheptanal;2,3,4,5,6-pentahydroxylhexanal; 2,3,4,5-tetrahydroxylhexanal; and2,3-dihydroxylpropanal.
 25. The method of claim 20, wherein saidhydrophilic polyol is glucose.
 26. The method of claim 20, wherein saidchitosan-derivative nanoparticle has an Arg final functionalizationdegree:HP final functionalization degree ratio between 1:1 to 10:1. 27.The method according to claim 26, wherein said Arg finalfunctionalization degree:HP final functionalization degree ratio isbetween 3:1 to 7:1.
 28. The method according to claim 28, wherein saidArg final functionalization degree:HP final functionalization degreeratio is 5:1.
 29. The method of claim 20, wherein the amine to phosphateratio of said DD-chitosan nucleic acid polyplex is between 2 to 100,between 2 to 50, between 2 to 30, or between 2 to
 15. 30. The method ofclaim 20, wherein said chitosan-derivative nanoparticle have an averagemolecular weight of less than 110 kDa before functionalization.
 31. Themethod of claim 20 wherein said nucleic acid is an artificial nucleicacid.
 32. The method of claim 31, wherein said artificial nucleic acidis selected from the group consisting of locked nucleic acid (LNA),glycol nucleic acid (GNA), and threose nucleic acid (TNA).
 33. Themethod of claim 20, wherein said nucleic acid is a therapeutic DNA orRNA.
 34. The method of claim 33, wherein said therapeutic RNA isselected from the group consisting of antisense RNA, siRNA, shorthairpin RNA, mRNA, micro RNA, or enzymatic RNA.
 35. The method of claim20, wherein said DD-chitosan nucleic acid polyplex has a pH selectedfrom a range consisting of between 3.0-8.0, between 4.0-7.0 or between4.5-6.5.
 36. The method of claim 20, wherein said DD-chitosan nucleicacid polyplex has an average polydispersity index (PDI) less than 0.5,less than 0.4, less than 0.3 or less than 0.25.
 37. The method of claim33 wherein said therapeutic DNA encodes a cytokine.
 38. The method ofclaim 33, wherein said therapeutic nucleic acid encodes insulin, aglucagon antagonist, GLP-1 or leptin.
 39. The method of claim 33,wherein said therapeutic nucleic acid encodes IL-10 or a TNFαantagonist.
 40. The method of claim 33, wherein said therapeutic nucleicacid encodes leptin, cholecystokinin, PYY or GLP-1.
 41. The method ofclaim 37 wherein said subject in need is a human patient suffering ahyperproliferative disorder in a urogenital tissue and the cytokine isIL-12.